Polynucleotides and their use for detecting resistance to streptogramin A or to streptogramin B and related compounds

ABSTRACT

The present invention pertains to polynucleotides derived from staphylococcal genes encoding resistance to streptogramin A or to streptogramin B and chemically related compounds. This invention relates to the use of polynucleotides as oligonucleotide primers or probes for detecting Staphylococcal strains that are resistant to streptogramin A or to streptogramin B and related compounds in a biological sample. The present invention is directed to the full length coding sequences of the staphylococcal genes encoding for resistance to streptogramin A or to streptogramin B from Staphylococcus and to polypeptides expressed by these full length coding sequences. This invention relates to the use of the expressed polypeptides to produce specific monoclonal or polyclonal antibodies that serve as detection means in order to characterize any staphylococcal strain carrying genes encoding resistance to streptogramin A or to streptogramin B.

This application is a 35 U.S.C. §371 application of Patent ApplicationPCT/IB98/00962, filed Jun. 22, 1998, which claims the benefit under 35U.S.C. 119(e) of the United States Provisional Application Serial No.60/050,380, filed Jun. 20, 1997, the entire disclosure of which isincorporated by reference herein.

The present invention pertains to polynucleotides derived fromstaphylococcal genes encoding resistance to streptogramin A or tostreptogramin B and chemically related compounds. This invention alsorelates to the use of the polynucleotides as oligonucleotide primers orprobes for detecting Staphylococcal strains that are resistant tostreptogramin A or to streptogramin B and related compounds in abiological sample.

In another embodiment, the present invention is directed to the fulllength coding sequences of the staphylococcal genes encoding forresistance to streptogramin A or to streptogramin B from Staphylococcusand to the polypeptides expressed by these full length coding sequences.

Further, this invention relates to the use of the expressed polypeptidesto produce specific monoclonal or polyclonal antibodies that serve asdetection means in order to characterize any staphylococcal straincarrying genes encoding resistance to streptogramin A or tostreptogramin B.

The present invention is also directed to diagnostic methods fordetecting specific strains of Staphylococcus expected to be contained ina biological sample. The diagnostic methods use the oligonucleotideprobes and primers as well as the antibodies of the invention.

Streptogramins and related compounds (antibiotics) produced bystreptomycetes can be classified as A and B compounds according to theirbasic primary structures (Cocito, 1979). Compounds of the A group,including streptogramin A (SgA), pristinamycin IIA (PIIA), virginiamycinM, mikamycin A, or synergistin A, are polyunsaturated cyclicmacrolactones Compounds of the B group, including streptogramin B (SgB),pristinamycin B (PIB), virginiamycin S, mikamycin B, and synergistin B,are cyclic peptidic macrolactones (Cocito, 1979). Compounds of bothgroups, A and B, bind different targets in the peptidyltransferasedomain of the 50S ribosomal subunit and inhibit protein elongation atdifferent steps (Aumercier et al., 1992; Di Giambattista et al., 1989).

A decrease in the dissociation constant of PIB is observed in thepresence of PIIA because this latter antibiotic provokes aconformational modification of the bacterial ribosome at the bindingsites of these molecules. Thus, A and B compounds, which arebacteriostatic when used separately, act synergistically when combinedand become bactericidal, mainly against Gram-positive bacteria.

Natural mixtures such as pristinamycin (Pt), synergistin, virginiamycinand mikamycin, are used orally and topically. A semi-syntheticinjectable streptogramin, RP59500, consisting of a mixture ofderivatives of A and B compounds (Dalfopristin and Quinupristin,respectively) is currently undergoing in vivo experimental and clinicaltrials (J. Antimicrob. Agents Chemother. 30 (Suppl. A), entire volume,1992; Entenza et al., 1995; Fantin et al., 1995; Griswold et al., 1996;Torralba et al., 1995). Staphylococcal resistance to synergisticmixtures of A and B compounds (Pt MIC≧2 μg/ml) is always associated withresistance to A compounds (PIIA MIC≧8 μg/ml), but not necessarily withresistance to B compounds (Allignet et al., 1996).

To date, four genes encoding resistance to A compounds have beenisolated from staphylococcal and enterococcal plasmids. The genes vat(Allignet et al., 1993), vatB (Allignet and El Solh, 1995), and satA(Rende-Fournier et al., 1993) encode related acetyltransferases(50.4-58.3% amino acids), which inactivate streptogramin A and similarcompounds. The staphylococcal gene vga (Allignet et al., 1992) encodesan ATP-binding protein probably involved in the active efflux of Acompounds. Nevertheless, there continues to exist a need in the art forpolynucleotides specific for Staphylococczis resistant to streptograminA and/or B and related compounds.

SUMMARY OF THE INVENTION

Accordingly, this invention aids in fulfilling this need in the art. Inparticular, this invention provides a purified peptide comprising anamino acid sequence selected from the group consisting of:

a) SEQ ID NO: 4 which corresponds to the complete amino acid sequence ofVga B or fragments derived from SEQ ID NO: 4 containing at least 10amino acids;

b) SEQ ID NO: 5 which corresponds to the complete amino acid sequence ofVat C or fragments derived from SEQ ID NO: 5 containing at least 10amino acids;

c) SEQ ID NO: 6 which corresponds to the complete amino acid sequence ofVgb B or fragments derived from SEQ ID NO: 6 containing at least 10amino acids;

d) SEQ ID NO: 7 which corresponds to the complete amino acid sequence ofVgb B;

e) SEQ ID NO: 8 which corresponds to a fragment of the amino acidsequence of Vga B;

f) SEQ ID NO: 9 which corresponds to a fragment of the amino acidsequence of Vat C; and

g) SEQ ID NO: 10 which corresponds to a fragment of the amino acidsequence of Vat C.

This invention additionally provides a purified polynucleotidecomprising the nucleotide sequence selected from the group consistingof:

a) SEQ ID NO: 1 which corresponds to the complete nucleic acid sequenceof vga B or fragments derived from SEQ ID NO: 1 containing 15 to 40nucleotides;

b) SEQ ID NO: 2 which corresponds to the complete nucleic acid sequenceof vat C or fragments derived from SEQ ID NO: 2 containing 15 to 40nucleotides;

c) SEQ ID NO: 3 which corresponds to the complete nucleic acid sequenceof vgb B or fragments derived from SEQ ID NO: 3 containing 15 to 40nucleotides;

d) SEQ ID NO: 11 which corresponds to the nucleic acid sequence encodingthe polypeptide of SEQ ID NO: 7;

e) SEQ ID NO: 12 which corresponds to the nucleic acid sequence encodingthe polypeptide of SEQ ID NO: 8;

f) SEQ ID NO: 13 which corresponds to the nucleic acid sequence encodingthe polypeptide of SEQ ID NO: 9; and

g) SEQ ID NO: 14 which corresponds to the nucleic acid sequence encodingthe polypeptide of SEQ ID NO: 10.

Furthermore, this invention includes a purified peptide comprising theamino acid sequence encoded by the nucleotide sequence selected from thegroup consisting of:

a) SEQ ID NO: 1,

b) SEQ ID NO: 2,

c) SEQ ID NO: 3,

d) SEQ ID NO: 11,

e) SEQ ID NO: 12,

f) SEQ ID NO: 13, and

g) SEQ ID NO: 14.

This invention also provides a composition comprising purifiedpolynucleotide sequences including at least one nucleotide sequenceselected from the group consisting of polynucleotides, genes or cDNA ofvgaB, vatC, and vgbB, which are useful for the detection of resistanceto streptogramin A and/or to streptogramin B and related compounds. Thisinvention further provides a composition comprising purified amino acidsequences including at least an amino acid sequence from a polypeptideencoded by a polynucleotide selected from the group consisting ofpolynucleotides, genes or cDNA of vgaB, vatC, and vgbB, which are usefulfor the detection of resistance to streptogramin A and/or tostreptogramin B and related compounds.

In another embodiment, this invention provides a composition ofpolynucleotide sequences encoding resistance to streptogramins andrelated compounds, or inducing this resistance in Gram-positivebacteria, wherein the composition comprises a combination of at leasttwo of the following, nucleotide sequences: a) a nucleotide sequenceencoding an acetyltransferase conferring resistance to streptogramin Aand related compounds, b) a nucleotide sequence encoding a moleculecontaining ATP binding motifs conferring resistance to streptogramin Aand related compounds; and c) a nucleotide sequence encoding a lactonaseconferring resistance to streptogramin B and related compounds.

Furthermore, this invention provides a composition of polynucleotidesequences, wherein the sequence encoding a molecule containing ATPbinding motifs confers resistance to Staphylococci and particularly toS. aureus, and wherein the polynucleotide sequence corresponds to a vgaBnucleotide sequence represented by SEQ ID NO: 1 or a sequence having atleast 70% homology with vgaB complete nucleotide sequence, or to apolynucleotide hybridizing with SEQ ID NO: 1 under stringent conditions,or to a fragment containing between 20 and 30 nucleotides of SEQ ID NO:11 or SEQ ID NO: 12, or wherein the polynucleotide sequence encodes apolypeptide having at least 60% homology with the complete SEQ ID NO: 4or with SEQ ID NO: 7 or SEQ ID NO: 8.

Furthermore this invention relates to a composition of polynucleotidesequences, wherein the sequence encoding an acetyltransferase confersresistance to streptogramin A and related compounds in Staphylococci,and particularly in S. cohnii, and wherein the polynucleotide sequencecorresponds to a vatC nucleotide sequence represented by SEQ ID NO: 2 ora sequence having at least 70% homology with vatC complete nucleotidesequence, or to a polynucleotide hybridizing with SEQ ID NO: 2 understringent conditions, or to a fragment containing between 20 and 30nucleotides of SEQ ID NO: 13 or SEQ ID NO: 14, or wherein thepolynucleotide sequence encodes a polypeptide having at least 60%homology with the complete SEQ ID NO: 5 or with SEQ ID NO: 9 or SEQ IDNO: 10.

This invention also provides a composition of polynucleotide sequences,wherein the sequence encoding a lactonase confers resistance tostreptogramin B and related compounds in Staphylococci and particularlyin S. cohnii, and wherein the polynucleotide sequence corresponds to avgbB nucleotide sequence represented in SEQ ID NO: 3 or a sequencehaving at least 70% homology with vgbB complete nucleotide sequence, orto a polynucleotide hybridizing with SEQ ID NO: 3 under stringentconditions, or to a fragment containing between 20 and 40 nucleotides ofSEQ ID NO: 3, or wherein the polynucleotide sequence encodes apolypeptide having at least 60% homology with the complete SEQ ID NO: 6.

The invention also contemplates a composition of polynucleotidesequences, wherein at least a vatB nucleotide sequence encoding anacetyltransferase conferring resistance to streptogramin A and relatedcompounds is included in addition to a vgaB nucleotide sequence encodinga molecule containing ATP binding motifs conferring resistance tostreptogramin A.

Additionally, the invention includes a purified polynucleotide thathybridizes specifically under stringent conditions with a polynucleotidesequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ IDNO: 14.

The invention further includes polynucleotide fragments comprising atleast 10 nucleotides capable of hybridization under stringent conditionswith any one of the nucleotide sequences enumerated above.

In another embodiment of the invention, a recombinant DNA sequencecomprising at least one nucleotide sequence enumerated above and underthe control of regulatory elements that regulate the expression ofresistance to antibiotics of the streptogramin family in a defined hostis provided.

Furthermore, the invention includes a recombinant vector comprising therecombinant DNA sequence noted above, wherein the vector comprises theplasmid pIP1633 or plasmid pIP1714.

The invention also includes a recombinant cell host comprising apolynucleotide sequence enumerated above or the recombinant vectordefined above.

In still a further embodiment of the invention, a method of detectingbacterial strains that contain the polynucleotide sequences set forthabove is provided.

Additionally, the invention includes kits for the detection of thepresence of bacterial strains that contain the polynucleotide sequencesset forth above.

The invention also contemplates antibodies recognizing peptide fragmentsor polypeptides encoded by the polynucleotide sequences enumeratedabove.

Still further, the invention provides for a screening method for activeantibiotics and/or molecules for the treatment of infections due toGram-positive bacteria, particularly staphylococci, based on thedetection of activity of these antibiotics and/or molecules on bacteriahaving the resistance phenotype to streptogramins.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

This invention will be more fully described with reference to thedrawings in which:

FIGS. 1A and 1B are the restriction maps of the 5.5 kb Bg/II fragmentand of the 2.4 kb HindIII-HaeIII fragment of pIP1633, respectively. Bothfragments confer resistance to streptogramin A and related compounds.The strategy for sequencing the 2.4 kb HindIII-HaeIII fragment is givenin FIG. 1B. Restriction enzyme abbreviations: Ba, BamHI; Bg, Bg/II; E,EcoRI; H, HindIII; X, XbaI.

FIG. 2 is the nucleotide sequence and deduced amino acid sequence of2411 nucleotides from pIP1633, which contains the gene vgaB of S. aureusconferring resistance to streptogramin A and related compounds. Theputative ribosome binding site (RBS) is underlined. The amino acids arealigned with the second nucleotide of each codon. Asterisks indicate thein-frame stop codons. The A and B ATP-binding motifs described by Walkeret al. (1982) and detected within each of the two ATP-domains are boxed.The conserved motif SGG of the two copies of loop 3 described by Hyde etal. (1990) is underlined. Relevant restriction sites are shown.

FIG. 3 is the amino acid sequence alignment of the predicted 60 and 61kDa proteins encoded by Vga (Allignet et al., 1992, accession No:m90056) and VgaB (FIG. 2), respectively. Identical residues areindicated by asterisks and conservative changes are shown by singledots. The A and B motifs of Walker et al. (1982) are in bold type (WA,WB). The conserved motif SGG of the two copies of loop 3 described byHyde et al. (1990) is underlined.

FIG. 4 is a restriction map of the plasmid pIP1714 carrying the genesvatC and vgbB as well as the genes pre and repB of S. cohnii strainBM10711 resistant to the synergistic mixtures of streptogramins A and B.

FIG. 5 is the nucleotide sequence and deduced amino acid sequence of1727 nucleotide from pIP1714, which contains the gene vgbB and vatC ofS. cohnii. Relevant restriction sites are shown.

FIGS. 6A, 6B, and 6C represent oligonucleotide primers for hybridizationunder stringent conditions with vatC, vgbB, and vgaB respectively.

FIG. 7 represents SEQ ID NOs: 1-14.

DETAILED DESCRIPTION OF THE INVENTION

It has now been determined that bacteria from the Staphylococcus genuscarry a vgaB gene, which encodes a putative ATP-binding protein thatconfers resistance to streptogramin A and structurally similarcompounds. It has also now been determined that bacteria from theStaphylococcus genus carry a vgbB gene, which encodes a lactonase thatconfers resistance to streptogramin B and structurally similarcompounds, and a vatC gene, which encodes an acetyltransferase thatconfers resistance to streptooramin A and structurally similarcompounds.

Novel polynucleotides corresponding to the vgaB, vgbB, and vatC genesfrom various strains of Staphylococcus have been isolated and sequenced,and it has been surprisingly demonstrated that these new polynucleotidesmake it possible to design oligonucleotide probes or primers. Thesepolynucleotides include the following:

a) SEQ ID NO: 1,

b) SEQ ID NO: 2,

c) SEQ ID NO: 3,

d) SEQ ID NO: 11,

e) SEQ ID NO: 12,

f) SEQ ID NO: 13, and

g) SEQ ID NO: 14.

This invention provides specific pairs of oligonucleotide primers orprobes that hybridize specifically, under stringent hybridizationconditions as defined hereinafter, to the nucleic acid (RNA or DNA) froma particular strain of the Staphylococcus genus. These oligonucleotideprimers include the following:

a)

Oligo I 5′-AAGTCGACTGACAATATGAGTGGTGG-3′ (SEQ ID NO: 17)

Oligo II 5′-CTGCAGATGCCTCAACAGCATCGATATCC-3′ (SEQ ID NO: 18)

b)

Oligo III 5′- ATGAATTCGCAAATCAGCAAGG-3′ (SEQ ID NO: 19)

Oligo IV 5′-TCGTCTCGAGCTCTAGGTCC-3′ (SEQ ID NO: 20)

c)

Oligo V 5′- CAGCAGTCTAGATCAGAGTGG-3′ (SEQ ID NO: 21)

Oligo VI 5′-CATACGGATCCACCTTTTCC-3′ (SEQ ID NO: 22)

In a specific embodiment of the present invention, the purifiedpolynucleotides useful for detecting Staphylococcal strains can be usedin combination in order to detect bacteria belonging to Staphylococci ina biological sample. Thus, the present invention also provides detectionmethods and kits comprising combinations of the purified polynucleotidesaccording to the invention. The purified oligonucleotides of theinvention are also useful as primers for use in amplification reactionsor as nucleic acid probes.

By “polynucleotides” according to the invention is meant the sequencesreferred to as SEQ ID NOs: 1, 2, 3, OR 11, 12, 13, 14 and thecomplementary sequences and/or the sequences of polynucleotides whichhybridize to the referred sequences in high stringent conditions andwhich are used for detecting staphylococcal strains carrying a geneencoding resistance to streptogramin A or to streptogramin B.

By “active molecule” according to the invention is meant a moleculecapable of inhibiting the activity of the purified polypeptide asdefined in the present invention or capable of inhibiting the bacterialculture of staphylococcal strains.

Thus, the polynucleotides of SEQ ID NOs: 1-3 and 11-14 and theirfragments can be used to select nucleotide primers notably for anamplification reaction, such as the amplification reactions furtherdescribed.

PCR is described in the U.S. Pat. No. 4,683,202 granted to Cetus Corp.The amplified fragments may be identified by agarose or polyacrylamidegel electrophoresis, or by a capillary electrophoresis, or alternativelyby a chromatography technique (gel filtration, hydrophobicchromatography, or ion exchange chromatography). The specificity of theamplification can be ensured by a molecular hybridization using asnucleic probes the polynucleotides of SEQ ID NOs: 1-3 and 11-14 andtheir fragments, oligonucleotides that are complementary to thesepolynucleotides or fragments thereof, or their amplification productsthemselves.

Amplified nucleotide fragments are useful as probes in hybridizationreactions in order to detect the presence of one polynucleotideaccording to the present invention or in order to detect the presence ofa bacteria of Staphylococcal strain carrying genes encoding resistanceto streptogramin A or streptogramin B, in a biological sample. Thisinvention also provides the amplified nucleic acid fragments(“amplicons”) defined herein above. These probes and amplicons can beradioactively or non-radioactively labeled, using for example enzymes orfluorescent compounds.

Preferred nucleic acid fragments that can serve as primers according tothe present invention are the following:

polynucleotides of sequence SEQ ID NOs: 1-3 and 11-14; and

polynucleotides having a length from 20 to 30 consecutive nucleotidesfrom a polynucleotide selected from the group consisting ofpolynucleotides of sequences SEQ ID NO: 11 to SEQ ID NO: 14 or from 20to 40 consecutive nucleotides from a polynucleotide of SEQ ID NO: 3.

The primers can also be used as oligonucleotide probes to specificallydetect a polynucleotide according to the invention.

Other techniques related to nucleic acid amplification can also be usedand are generally preferred to the PCR technique. The StrandDisplacement Amplification (SDA) technique (Walker et al., 1992) is anisothermal amplification technique based on the ability of a restrictionenzyme to cleave one of the strands at a recognition site (which isunder a hemiphosphorothioate form), and on the property of a DNApolymerase to initiate the synthesis of a new strand from the 3′ OH endgenerated by the restriction enzyme and on the property of this DNApolymerase to displace the previously synthesized strand being localizeddownstream.

The SDA amplification technique is more easily performed than PCR (asingle thermostated water bath device is necessary), and is faster thanthe other amplification methods. Thus, the present invention alsocomprises using the nucleic acid fragments according to the invention(primers) in a method of DNA or RNA amplification according to the SDAtechnique. The polynucleotides of SEQ ID NOs: 1-3 and 11-14 and theirfragments, especially the primers according to the invention, are usefulas technical means for performing different target nucleic acidamplification methods such as:

TAS (Transcription-based Amplification System), described by Kwoh et al.in 1989;

SR (Self-Sustained Sequence Replication), described by Guatelli et al.in 1990;

NASBA (Nucleic acid Sequence Based Amplification), described by Kievitiset al. in 1991; and

TMA (Transcription Mediated Amplification).

The polynucleotides of SEQ ID NOs: 1-3 and 11-14 and their fragments,especially the primers according to the invention, are also useful astechnical means for performing methods for amplification or modificationof a nucleic acid used as a probe, such as:

LCR (Ligase Chain Reaction), described by Landegren et al. in 1988 andimproved by Barany et al. in 1991, who employ a thermostable ligase;

RCR (Repair Chain Reaction), described by Segev et al. in 1992;

CPR (Cycling Probe Reaction), described by Duck et al. in 1990; and

Q-beta replicase reaction, described by Miele et al. in 1983 andimproved by Chu et al. in 1986, Lizardi et al. in 1988, and by Burg etal. and Stone et al. in 1996.

When the target polynucleotide to be detected is RNA, for example mRNA,a reverse transcriptase enzyme can be used before the amplificationreaction in order to obtain a cDNA from the RNA contained in thebiological sample. The generated cDNA can be subsequently used as thenucleic acid target for the primers or the probes used in anamplification process or a detection process according to the presentinvention.

Nucleic probes according to the present invention are specific to detecta polynucleotide of the invention. By “specific probes” according to theinvention is meant any oligonucleotide that hybridizes with onepolynucleotide of SEQ ID NOs: 1-3 and 11-14 and which does not hybridizewith unrelated sequences. Preferred oligonucleotide probes according tothe invention are oligonucleotides I-VI.

In a specific embodiment, the purified polynucleotides according to thepresent invention encompass polynucleotides having at least 80% homologyin their nucleic acid sequences with polynucleotides of SEQ ID NO: 11 toSEQ ID NO: 14, at least 70% identity with SEQ ID NO: 1 to 3. Bypercentage of nucleotide homology according to the present invention isintended a percentage of identity between the corresponding bases of twohomologous polynucleotides, this percentage of identity being purelystatistical and the differences between two homologous polynucleotidesbeing located at random and on the whole length of said polynucleotides.

The oligonucleotide probes according to the present invention hybridizespecifically with a DNA or RNA molecule comprising all or part of onepolynucleotide among SEQ ID NOs: 1-3 and 11-14 under stringentconditions. As an illustrative embodiment, the stringent hybridizationconditions used in order to specifically detect a polynucleotideaccording to the present invention are advantageously the following:

Prehybridization and hybridization are performed at 68° C. in a mixturecontaining:

5×SSPE (1×SSPE is 0.3 M NaCl, 30 mM tri-sodium citrate

5×Denhardt's solution

0.5% (w/v) sodium dodecyl sulfate (SDS); and

100 μg ml⁻¹ salmon sperm DNA

The washings are performed as follows:

Two washings at laboratory temperature for 10 min. in the presence of2×SSPE and 0.1% SDS;

One washing at 68° C. for 15 min. in the presence of 1×SSPE, 1% SDS; and

One washing at 68° C. for 15 min. in the presence of 0.1×SSPE and 0.1%SDS.

The non-labeled polynucleotides or oligonucleotides of the invention canbe directly used as probes. Nevertheless, the polynucleotides oroligonucleotides are generally labeled with a radioactive element (³²P,³⁵S, ³H, ¹²⁵I) or by a non-isotopic molecule (for example, biotin,acetylaminofluorene, digoxigenin, 5-bromodesoxyuridin, fluorescein) inorder to generate probes that are useful for numerous applications.Examples of non-radioactive labeling of nucleic acid fragments aredescribed in the French Patent No. FR 78 10975 or by Urdea et al. orSanchez-Pescador et al. 1988.

Other labeling techniques can also be used, such as those described inthe French patents 2 422 956 and 2 518 755. The hybridization step maybe performed in different ways (Matthews et al. 1988). A general methodcomprises immobilizing the nucleic acid that has been extracted from thebiological sample on a substrate (nitrocellulose, nylon, polystyrene)and then incubating, in defined conditions, the target nucleic acid withthe probe. Subsequent to the hybridization step, the excess amount ofthe specific probe is discarded, and the hybrid molecules formed aredetected by an appropriate method (radioactivity, fluorescence, orenzyme activity measurement).

Advantageously, the probes according to the present invention can havestructural characteristics such that they allow signal amplification,such structural characteristics being, for example, branched DNA probesas those described by Urdea et al. in 1991 or in the European Patent No.0 225 807 (Chiron).

In another advantageous embodiment of the present invention, the probesdescribed herein can be used as “capture probes”, and are for thispurpose immobilized on a substrate in order to capture the targetnucleic acid contained in a biological sample. The captured targetnucleic acid is subsequently detected with a second probe, whichrecognizes a sequence of the target nucleic acid that is different fromthe sequence recognized by the capture probe.

The oligonucleotide fragments useful as probes or primers according tothe present invention can be prepared by cleavage of the polynucleotidesof SEQ ID NOs: 1-3 and 11-14 by restriction enzymes, as described inSambrook et al in 1989. Another appropriate preparation process of thenucleic acids of the invention containing at most 200 nucleotides (or200 bp if these molecules are double-stranded) comprises the followingsteps:

synthesizing DNA using the automated method ofbetacyanethylphosphoramidite described in 1986;

cloning the thus obtained nucleic acids in an appropriate vector; and

purifying the nucleic acid by hybridizing to an appropriate probeaccording to the present invention.

A chemical method for producing the nucleic acids according to theinvention, which have a length of more than 200 nucleotides (or 200 bpif these molecules are double-stranded), comprises the following steps:

Assembling the chemically synthesized oligonucleotides having differentrestriction sites at each end;

cloning the thus obtained nucleic acids in an appropriate vector; and

purifying the nucleic acid by hybridizing to an appropriate probeaccording to the present invention.

The oligonucleotide probes according to the present invention can alsobe used in a detection device comprising a matrix library of probesimmobilized on a substrate, the sequence of each probe of a given lengthbeing localized in a shift of one or several bases, one from the other,each probe of the matrix library thus being complementary to a distinctsequence of the target nucleic acid. Optionally, the substrate of thematrix can be a material able to act as an electron donor, the detectionof the matrix positions in which hybridization has occurred beingsubsequently determined by an electronic device. Such matrix librariesof probes and methods of specific detection of a target nucleic acid aredescribed in the European patent application No. 0 713 016, or PCTApplication No. WO 95 33846, or also PCT Application No. WO 95 11995(Affymax Technologies), PCT Application No. WO 97 02357 (AffymetrixInc.), and also in U.S. Pat. No. 5,202,231 (Drmanac), said patents andpatent applications being herein incorporated by reference

The present invention also pertains to a family of recombinant plasmidscontaining at least a nucleic acid according to the invention. Accordingto an advantageous embodiment, a recombinant plasmid comprises apolynucleotide of SEQ ID NOs: 1-3 and 11-14 or one nucleic fragmentthereof. More specifically, the following plasmids are part of theinvention: pIP1633 and pIP1714.

The present invention is also directed to the full length codingsequences of the vgaB, vgbB, and vatC genes from Staphylococci that areavailable using the purified polynucleotides according to the presentinvention, as well as to the polypeptide enzymes encoded by these filllength coding sequences. In a specific embodiment of the presentinvention, the full length coding sequences of the vgaB, vgbB, and vatCgenes are isolated from a plasmid or cosmid library of the genome ofStaphylococci that have been screened with the oligonucleotide probesaccording to the present invention. The selected positive plasmid orcosmid clones hybridizing with the oligonucleotide probes of theinvention are then sequenced in order to characterize the correspondingfull length coding sequence, and the DNA insert of interest is thencloned in an expression vector in order to produce the corresponding ATPbinding motif conferring resistance to streptogramin A and relatedcompounds, acetyltransferase conferring resistance to streptogramin Aand related compounds, or lactonase conferring resistance tostreptogramin B and related compounds.

A suitable vector for the expression in bacteria and in particular in E.coli, is the pQE-30 vector (QIAexpress) that allows the production of arecombinant protein containing a 6×His affinity tag. The 6×His tag isplaced at the C-terminus of the recombinant polypeptide ATP bindingmotif conferring resistance to streptograrnin A and related compounds,acetyltransferase conferring resistance to streptogramin A and relatedcompounds or lactonase conferring resistance to streptogramin B andrelated compounds, which allows a subsequent efficient purification ofthe recombinant polypeptide ATP binding motif conferring resistance tostreptogramin A and related compounds, acetyltransferase conferringresistance to streptogramin A and related compounds, or lactonaseconferring resistance to streptogramin B and related compounds bypassage onto a nickel or copper affinity chromatography column. Thenickel chromatography column can contain the Ni-NTA resin (Porath et al.1975).

The polypeptides according to the invention can also be prepared byconventional methods of chemical synthesis, either in a homogenoussolution or in solid phase. As an illustrative embodiment of suchchemical polypeptide synthesis techniques the homogenous solutiontechnique described by Houbenweyl in 1974 may be cited.

The polypeptides according to the invention can be characterized bybinding onto an immunoaffinity chromatography column on which polyclonalor monoclonal antibodies directed to a polypeptide among the ATP bindingmotif conferring resistance to streptogramin A and related compounds,acetyltransferase conferring resistance to streptogramin A and relatedcompounds, or lactonase conferring resistance to streptogramin B andrelated compounds of the invention have previously been immobilized.

Another object of the present invention comprises a polypeptide producedby the genetic engineering techniques or a polypeptide synthesizedchemically as above described.

The polypeptide ATP binding motif conferring resistance to streptograminA and related compounds, acetyltransferase conferring resistance tostreptogramin A and related compounds, or lactonase conferringresistance to streptogramin B and related compounds according to thepresent invention are useful for the preparation of polyclonal ormonoclonal antibodies that recognize the polypeptides or fragmentsthereof. The monoclonal antibodies can be prepared from hybridomasaccording to the technique described by Kohler and Milstein in 1975. Thepolyclonal antibodies can be prepared by immunization of a mammal,especially a mouse or a rabbit, with a polypeptide according to theinvention that is combined with an adjuvant, and then by purifyingspecific antibodies contained in the serum of the immunized animal on aaffinity chromatography column on which has previously been immobilizedthe polypeptide that has been used as the antigen.

Consequently, the invention is also directed to a method for detectingspecifically the presence of a polypeptide according to the invention ina biological sample. The method comprises:

a) bringing into contact the biological sample with an antibodyaccording to the invention; and

b) detecting antigen-antibody complex formed.

Also part of the invention is a diagnostic kit for in vitro detectingthe presence of a polypeptide according to the present invention in abiological sample. The kit comprises:

a polyclonal or monoclonal antibody as described above, optionallylabeled; and

a reagent allowing the detection of the antigen-antibody complexesformed, wherein the reagent carries optionally a label, or being able tobe recognized itself by a labeled reagent, more particularly in the casewhen the above-mentioned monoclonal or polyclonal antibody is notlabeled by itself.

Indeed, the monoclonal or polyclonal antibodies according to the presentinvention are useful as detection means in order to identify orcharacterize a Staphylococcal strain carrying genes encoding resistanceto streptogramin A or streptogramin B.

The invention also pertains to:

A purified polypeptide or a peptide fragment having at least 10 aminoacids, which is recognized by antibodies directed against apolynucleotide sequence conferring resistance to streptogramin andrelated compounds, corresponding to a polynucleotide sequence accordingto the invention.

A polynucleotide comprising the full length coding sequence of aStaphylococcus streptogramin A and/or B resistant gene containing apolynucleotide sequence according to the invention.

A monoclonal or polyclonal antibody directed against a polypeptide or apeptide fragment encoded by the polynucleotide sequences according tothe invention.

A method of detecting the presence of bacterium harboring thepolynucleotide sequences according to the invention in a biologicalsample comprising:

a) contacting bacterial DNA of the biological sample with a primer or aprobe according to the invention, which hybridizes with a nucleotidesequence encoding resistance to streptogramins;

b) amplifying the nucleotide sequence using said primer or said probe;and

c) detecting the hybridized complex formed between said primer or probewith the DNA.

A kit for detecting the presence of bacterium having resistance tostreptogramin A and/or streptogramin B and harboring the polynucleotidesequences according to the invention in a biological sample, said kitcomprising:

a) a polynucleotide probe according to the invention; and

b) reagents necessary to perform a nucleic acid hybridization reaction.

A kit for detecting the presence of bacterium having resistance tostreptogramin A and harboring the polynucleotide sequences according tothe invention in a biological sample, said kit comprising:

a) a polynucleotide probe according to the invention; and

b) reagents necessary to perform a nucleic acid hybridization reaction.

A method of screening active antibiotics for the treatment of theinfections due to Gram-positive bacteria, comprising the steps of:

a) bringing into contact a Gram-positive bacteria having a resistance tostreptogramin A or streptogramin B and related compounds and containingthe polynucleotide sequences according to the invention with theantibiotic; and

b) measuring an activity of the antibiotic on the bacteria having aresistance to streptogramins and related compounds.

A method of screening for active synthetic molecules capable ofpenetrating into a bacteria of the family of staphylococci, wherein theinhibiting activity of these molecules is tested on at least apolypeptide encoded by the polynucleotide sequences according to theinvention comprising the steps of:

a) contacting a sample of said active molecules with the bacteria;

b) testing the capacity of the active molecules to penetrate into thebacteria and the capacity of inhibiting a bacterial culture at variousconcentration of the molecules; and

c) choosing the active molecule that provides an inhibitory effect of atleast 80% on the bacterial culture compared to an untreated culture.

An in vitro method of screening for active molecules capable ofinhibiting a polypeptide encoded by the polynucleotide sequencesaccording to the invention, wherein the inhibiting activity of thesemolecules is tested on at least said polypeptide, said method comprisingthe steps of:

a) extracting a purified polypeptide according to the invention;

b) contacting the active molecules with said purified polypeptide;

c) testing the capacity of the active molecules, at variousconcentrations, to inhibit the activity of the purified polypeptide; and

d) choosing the active molecule that provides an inhibitory effect of atleast 80% on the activity of the said purified polypeptide.

A composition of a polynucleotide sequence encoding resistance tostreptogramins and related compounds, or inducing resistance inGram-positive bacteria, wherein said composition comprises a nucleotidesequence corresponding to the resistance phenotype of the plasmidpIP1633 deposited with the C.N.C.M. under the Accession No. I-1768 andof the plasmid pIP1680 deposited with the C.N.C.M. under the AccessionNo. I-1767 and of the plasmid pIP1714 deposited with the C.N.C.M. underthe number I-1 877 on Jun. 18, 1997.

A method of detecting the presence of bacterium harboring thepolynucleotide sequences according to the invention in a biologicalsample, said method comprising the steps of:

a) contacting said sample with an antibody according to the inventionthat recognizes a polypeptide encoded by said polynucleotide sequences;and

b) detecting said complex.

A diagnostic kit for in vitro detecting the presence of bacteriumharboring the polynucleotide sequences according to the invention in abiological sample, said kit comprising:

a) a predetermined quantity of monoclonal or polyclonal antibodiesaccording to the invention;

b) reagents necessary to perform an immunological reaction between theantibodies and a polypeptide encoded by said polynucleotide sequences;and

c) reagents necessary for detecting said complex between the antibodiesand the polypeptide encoded by said polynucleotide sequences.

The inhibiting activity of the molecules can be readily evaluated by oneskilled in the art. For example, the inhibiting activity of Vga B can betested by detecting its ATP hydrolysis as described in J. I. Ross et al.(1990), Mol. Microbiol. 4(7):1207-1214 regarding the rate evaluation ofthe active efflux of antibiotics from a cell. Ross et al. use adifferent gene, but their gene product functions as a drug efflux pumpin the same way as Vga B does.

The inhibiting activity of Vat C can be tested by visualizing theacetylation reaction as described in Allignet et al. (1993) regardingthe mechanism of inactivation of A-type compounds conferred by plasmidspIP680 and pIP1156 by thick layer chromatography and NMR.

The inhibiting activity of Vgb B can be tested by detecting thedegradation of streptogramin B or a related compound by amicrobiological test as described in Allignet et al. (1988).

Plasmids containing the polynucleotides from Staphylococci, which conferstreptogramin A and/or B resistance, are referred to herein by thefollowing accession numbers:

Plasmid Accession No pIP1714 I-1877 pIP1633 I-1768 pIP680 I-1767

and they have been inserted into vectors which have been deposited atthe Collection Nationale de Cultures de Microorganismes (“C.N.C.M.”)Institut Pasteur, 28, rue du Docteur Roux, F-75724 Paris Cedex 15,France on Jun. 18, 1997, and Aug. 7, 1996, respectively.

EXAMPLES Example 1 Cloning of the vgaB Gene Carried by Plasmid pIP1633

pPI633 was isolated from a S. aureus transconjugant strain, BM12235,obtained from the donor wild-type S. aureus strain, BM3385 (Allignet andEl Solh, 1995). This plasmid carried the vatB gene located on a 5.5BglII fragment, but the other described streptogramin A resistant(SgA^(r)) genes were not detected either by hybridization experiments orby PCR (Allignet and EI Solh, 1995). Since the gene vga was carried byall the tested staphylococcal plasmids containing the vat gene (Allignetet al., 1996), the presence of a vga-related gene was suspected inpIP1633. We therefore searched this gene in the recombinant plasmid,pIP1675 (FIG. 1A), containing the vatB-5.5 BglII fragment of pIP1633.

First, the 2.4 kb HindIII-HaeIII fragment of pIP 1675, which containsonly 10 nucleotide from vatB, was inserted into plasmid pOX300, and therecombinant plasmid, pIP1717 (FIG. 1B), was introduced byelectroporation into the S. aureus recipient, RN4220 (Kreiswirth et al.,1983). Plasmid pOX300, also named pOX7, (Dyke and Curnock, 1989), is ahybrid of pUC18 and pE194ts and replicates in E. coli where it confersresistance to ampicillin and to erythromycin, and in S. aureus whereonly resistance to erythromycin is expressed. The S. aureustransformants selected on 10 μg/ml erythromycin were resistant tostreptogramin A and related compounds (PIIA MICs=8-16 μg/ml). Thus, the2.4-kb HindIII-HaeIII insert of pIP1717 (FIG. 1B) probably carried astreptogramin A resistance gene and was sequenced. The nucleotide(nucleotide) sequence of this fragment was determined by the dideoxymethod (Sanger et al., 1977) with the reagents and the procedurerecommended by the suppliers of the T⁷ sequencing kit (PharmaciaInternational). Arrows indicate the direction and extent of eachdideoxy-sequencing reaction. (FIG. 1B).

Example 2 The Nucleotide Sequence of the vgaB Gene

The strategy of sequencing on both strands is outlined in FIG. 1 and thesequence of the 2411-bp HindIII-HaeIII insert is given in FIG. 2. Anopen reading frame (ORF) of 1674 nucleotide extending from nucleotide682 to 2356 was detected on the same strand as vatB (FIG. 2). The 1674nucleotide ORF contained an ATG start codon at nucleotide 700 to 702 andwas preceded by an 8 nucleotide putative RBS. The ΔG (free energy ofassociation) of interaction of the most stable structure between thisputative RBS and the 3′-terminus of the 165 rRNA (MacLaughlin et al.,1981; Moran et al., 1982) calculated according to Tinoco et al. (1973)was −79.4 kJ/mol. The sequence located between the ATG codon and the TAAstop codon at nucleotide 2356 to 2358 may encode a 552 amino acidprotein of 61,327 daltons (Da). This putative gene, named vgaB, had58.8% nucleotide identity with the 1572 bp gene, vga (Allignet et al.,1992). The G+C content of vgaB (27.2%) is similar to that of vga (29%),but both values are slightly lower than those of the staphylococcalgenome (32 to 36%) (KIoos and Schleifer, 1986). The nucleotide sequenceof vgaB has been submitted to the GenBank/EMBL data bank under accessionno. u82085.

Example 3 Amino Acid Sequence Analysis of VgaB

The predicted translation product of the vgaB gene, VgaB, has acalculated isoelectric point (pI) of 9.60. The hydropathy plot of theVgaB sequence according to the algorithm of Kyte and Doolittle (1982)indicates the protein to be hydrophilic. No similarity to known signalsequences of secreted proteins (von Heijne, 1986; Watson, 1984) wasobserved.

The amino acid sequence of VgaB was compared with the sequencesavailable in databases (GenBank, release 97.0; EMBL, release 48;SwissProt, release 34). Significant similarity to the ATP-bindingdomains of numerous ATP-binding Cassette (ABC) proteins was found. Theprotein giving the best match was Vga (48.3% identical amino acid, 70.4%similar amino acid). VgaB and Vga each contain two ATP-binding domainssharing 38.8% and 39.1% identical amino acid, respectively. Each ofthese domains includes the two ATP-binding motifs described by Walker etal. (1982) (FIG. 2). Moreover, the highly conserved SGG sequence of loop3 found between the two ATP-binding motifs of all investigatedATP-binding, proteins (Barrasa et al., 1995; Hyde et al., 1990) wasdetected in Vga (Allignet et al., 1992) and VgaB (FIG. 2). According tothe predicted tertiary structure of ABC model cassette, this loop wouldbe conveniently located to interact with the cell membrane (Hyde et al.,1990). The inter-ATP-binding domain of VgaB is more rich in glutamine(11 Q in 155 amino acid total) than the rest of the sequence of theprotein (11 Q/397 amino acid). In contrast, the proportion of glutaminein the inter-ATP-binding domain of Vga is similar to that in the otherpart of the protein (4 Q/156 amino acid and 14 Q/366 amino acid,respectively). Neither Vga nor VgaB contains hydrophobic transmembranedomains.

The ABC protein MsrA (Ross et al., 1990) is the most similar to Vga andVgaB (35.2% and 34.4% identical amino acid, respectively). MsrA confersresistance to erythromycin by increasing the efflux of this antibioticand to streptogramin B by a mechanism not yet elucidated. MsrA containstwo ATP-binding domains with 31.8% amino acid identity and separated bya Q-linker, but no hydrophobic stretches that might be potentialmembrane spanning domains. The hydrophobic proteins, which are expectedto interact with MsrA, are those encoded by similar genes mapping nearMsrA in two staphylococcal strains (smpA, smpB) and also those on thechromosome of the S. aureus recipient strain, RN4220 (smpC), which doesnot carry msrA (Ross et al., 1995). Ross et al. (1996) have recentlyreported that SmpC found in the chromosome of RN4220 is not essentialfor the expression of resistance to erythromycin conferred by MsrA.Thus, further experiments are required to elucidate the mechanisms ofresistance conferred by msrA, vga, or vgaB genes.

Several ABC transporters, which do not have alternating hydrophobicdomains, have been grouped in a subfamily in order to distinguish themfrom the members of the ABC₂ transporter subfamily, the members of whichcontain hydrophobic transmembrane domains (Barrasa et al., 1995; Olanoet al., 1995; Peschke et al., 1995). Thus, VgaB may be considered as anew member of the former ABC transporter subfamily. Excluding VgaB, Vga,and MsrA, most of the known ABC transporters that contain twoATP-binding cassettes but no hydrophobic domain(s) were found inantibiotic or antibiotic producing microorganisms in which they areinvolved in the active excretion of these molecules. These transportersare encoded by the following genes: ardI, an amino-acylnucleosideantibiotic resistance gene from Streptomyces capreolus (Barrasa et al.,1995); carA, a carbomycin-resistance gene from Streptomycesthermotolerans (Schoner et al., 1992); ImrC, a lincomycin-resistancegene from Streptomyces lincolnensis (Peschke et al., 1995); oleB, anoleandomycin-resistance gene from Streptomyces antibioticus (Olano etal., 1995); srmB, a spiramycin-resistance gene from Streptomycesambofaciens (Geistlich et al., 1992); tlrC, a tylosin-resistance genefrom Streptomyces fradiae (Rosteck et al., 1991); and petT, a pep5epidermin-resistance gene from Staphylococcus epidermidis (Meyer et al.,1995). The amino acid identity between each of these latter ABCtransporters and VgaB is between 23.6% and 28.7%.

Degenerate primers designed from an analysis of the alignment of theamino acid sequence of Vga and VgaB may be helpful to detect suchputative genes by PCR experiments. In the streptogramins producers, thedescribed resistance to these antibiotics consists of streptogramin Ainactivation by an as yet unknown mechanism (Fierro et al., 1989),streptogramin B inactivation by a lactonase (Kim et al., 1974) andputative increased export of streptogramin A and streptogramin B by anintegral membrane protein, Ptr, exploiting transmembrane protongradients (Blanc et al., 1995). The NMR spectra of the modified Acompounds may be analyzed to verify if their inactivation in theantibiotic producers is similar to that due to the proteins Vat or VatB,which transfer an o-acetyl group to position C14 of PIIA (Allignet etal., 1993). Interestingly, the staphylococcal gene vgb (Allignet et al.,1988) found in most plasmids carrying vga and vat (Allignet et al.,1996), encodes a protein inactivating streptogramin B and relatedcompounds by cleavage of the lactone ring.

Example 4 Distribution and Location of the vgaB Gene in 52 SgA^(R) andIndependent Wild-type Staphylococci

A recombinant plasmid containing a fragment of vgaB, pIP1705, wasconstructed to serve as a probe in hybridization experiments understringent conditions as described previously (Allignet et al., 1996).pIP1705 consists of pUC19 cleaved with Sall and PstI, and an insert of1051 bp amplified from within vgaB by the following primers, whichintroduce PstI or SalI sites:

(SEQ ID NO: 17) Oligo I 5′-AA GTCGAC TGACAATATGAGTGGTGG-3′       SalI(SEQ ID NO: 18) Oligo II 5′- CTGCAG ATGCCTCAACAGCATCGATATCC-3′      PstI

The 52 SgA^(r) staphylococci investigated (Allignet et al., 1996; ElSolh et al., 1980; Loncle et al., 1993) included 10 strains (7S. aureus,1S. simulans, 1S. haemolyticus, and 1S. cohnii urealyticum), whichharbored 26 to 45 kb plasmids containing vga, vat, and vgb; 21 strains(20 S. aureus and one S. epidermidis), which harbored 50 to 90 kbplasmids containing vatB; 16 strains (12 S. epidermidis, three S.haemolyticus and one S. aureus) with 6 to 15 kb plasmids containing vga;one S. epidermidis strain which harbored a plasmnid of approximately 20kb containing vga-vat; and four S. aureus strains, which do not carrynucleotide sequences hybridizing with vat, vatB, vga, or vgb. Nucleotidesequences hybridizing with pIP1705 were found only in the 21 largeplasmids containing vatB. In all these 21 plasmids including pIP1633,the hybridizing nucleotide sequences were detected on a 1.5 kb EcoRIfragment, which also hybridized with vatB, suggesting that vgaB and vatBhave conserved relative positions.

Example 5 Results Concerning vatC and vgbB Genes

The Staphylococcus cohnii strain, BM10711, resistant to the synergisticmixtures streptogramin A and streptogramin B and related compounds(pristinamycin, virginiamycin, synergistin, mikamycin,Quinupristin-Dalfopristin) was analyzed. This strain was isolated atDouera hospital (Algeria) where the pristinamycin was frequently usedtopically. The strain was isolated (Liassin et al., 1997) from a sampleprovided from a cupboard located in a room occupied by patientssuffering from chronic osteomyelitis.

The strain BM10711 harbored several plasmids including pIP1714 (5kb).This plasmid was isolated by electroporation in a S. aureus recipientstrain, RN4220. The transformant, harboring pIP1714, was selected onBHIA containing 10 μg/ml pristinamycin IIA. Plasmid pIP1714 conferredresistances to streptogramin A and streptogramin B and relatedcompounds.

Plasmid pIP1714 was linearized by cleavage with HindIII and cloned inthe HindIII site of the vector pOX7 also named pOX300 (Dyke et al.,1989, FEMS Microbiol. Lett. 58:209-216). pOX7 results from thecointegrationof the E. coil vector, pUC18, and S. aureus plasmid, pE194.The recombinant plasmid pIP1715 consisting of pOX7 and pIP1714 was usedto sequence pIP1714 in its entirety. The gene vatC (636 nucleotides)encoding an acetyltransferase inactiving streptogramin A and relatedcompounds and the gene vgbB (885 nucleotides) encoding a lactonaseinactiving streptogramin B and related compounds were found to becarried by this plasmid. The gene vatC had 71.7, 62.2 and 64.1%nucleotides identity with vat-related gene, vatB and satA respectivelyand the gene vgbB presents 69.5% nucleotides identity with the gene vgb.

VatC acetyltransferase exhibits significant similarity withacetyltransferases having the same enzymatic activity and encoded by thegenes vatC, vatB, and sat (respectively 69.8, 58.2 and 66.0% amino acidsidentity). These proteins belong to a family of xenobioticacetyltransferases modifying various substrates including streptograminA and related antibiotics. VgbB lactonase exhibits as well significantsimilarity with Vgb inactivating streptogramin B and related (67.0%amino acids identity).

The two other genes carried by pIP1714 are pre and repB, encodingproteins involved in mobilization and replication, respectively. Thesetwo genes are homologous to those carried by the staphylococcal plasmid,pUB 110 (McKenzie et at., 1986, Plasmid 15:93-103). Moreover, asreported in FIG. 5, the intergenic sequences of pIP1714 delimited byvatC and repB also exhibited significant similarities with pUB110.

Example 6 Plasmid DNA Isolation from PIIA^(R) Staphylococci

The staphylococci were grown after overnight incubation at 37° C. in 200ml BHI containing 10 μg/ml of PIIA. After 15 min centrifigation at 8000rpm, the pellet was resuspended in 25 ml TES (Tris 50 mM, EDTA 1 mM,saccharose 7%). After adding 150 μg of lysostaphin, the mixture wasincubated 30 min at 37° C. Then, 2ml of SDS 20% and 6 ml of EDTA 0.25 Mwere added and the suspension was incubated 15 min at 37° C. 8 ml ofNaCl 5M were added and the mixture was kept 90 min at +4° C. After 30min centrifugation at 8000 rpm, the supernatant was incubated 15 min at37° C. with 5 μg of Rnase (Boehringer). 10 μg of Proteinase K were addedand the suspension was incubated 15 min at 65° C. DNA was precipitatedusing isopropanol (0.6 V for 1 V of DNA solution). After 30 mincentrifugation at 8000 g, the pellet was washed with 10 ml ethanol 70%.The washed DNA was dried at 56° C., dissolved in 10 ml water andpurified by dye-buoyant density centrifugation (ethidium bromide-cesiumchloride). The extrachromosomal band was collected. After removingethidium bromide, the solution of plasmid DNA was dialysed using TEbuffer (Tris, 10 mM, EDTA 1 mM, pH 7).

Example 7 Plasmid DNA Isolation from E. coli

Cf. QIAfilter plasmid maxi protocol for large-scale preparations andQIAprep Spin plasmid kit protocol for mini-preparations.

Quiagen GmbH and Quiagen Inc. (Hilden, Germany)

Plasmid maxi kit Ref : 12262 Miniprep kit Ref : 27104

Example 8 Transformation by Electroporation of the S. aureus RecipientStrain, RN4220

1—Preparation of Cells 200 ml of BHI was inoculated with 20 ml of anovernight culture of RN4220 (Kreiswirth et al., Nature 1983,306:709-712) and incubated at 37° C. with shaking. When the OD reached0.4 at 600 nm, the suspension was kept in ice. The pellet was washedthree times with 20 ml of cold Hepes buffer (saccharose 9.31%-Hepes0.19%-pH. 7.4). The pellet was resuspended in 2.5 ml of Hepes buffercontaining 10% glycerol. Aliquots of 100 μl cell suspension (3.10¹⁰/ml)were stored at −80° C.

2—Electroporation

After thawing at room temperature, the 100 μl aliquot of cells was keptin ice. After adding 10 μl of a solution containing 1 μg of plasmid DNA,the mixture was transferred to a cold 0.2 cm electroporation cuvette.The Gene Pulser (BioRad) was set at 25 uF and 2.5 KV and the PulseController to 100Ω. This produced a pulse with a constant time of 2.3 to2.5 m sec. The cuvette was removed from the chamber and 1 ml of SOC (2%bactotryptone, 0.5% bactoyeast extract, 10 mM NaCl, 2.5 mMKCl, 10 mMMgCl₂, 10 mM MgSO₄, 20 mM glucose) was added. The cell suspension wastransferred in a propylene tube and incubated with shaking at 37° C. forI hr. The suspension was then plated on selective medium, whichconsisted of BHIA containing 10 μg/ml erythromycin or 10 μg/ml of PIIA.The plates were incubated 48 h at 37° C. and the transformants isolatedon selective medium. The further studies were carried out on a singleisolated colony.

Example 9 Polymerase Chain Reaction

DNA was amplified by PCR in a Crocodile II thermal cycler (Appligene)with approximately 10 ng of cellular DNA or 1 ng of plasmid DNA. Thereaction mixture contained 0.6 μM of each oligonucleotide serving asprimer, 200 μM of each deoxynucleotide triphosphate, 2.5 U of Taq DNAPolymerase (Amersham, Int.), and 1×buffer (Amersham, Int.). The finalreaction volume was adjusted to 100 μl with H₂O and the sample was thencovered by 50 μl of heavy white mineral oil (Sigma Chemical Co, St.Louis, Mo.).

PCR experiments were carried out at high or low stringency, depending onthe primers used. At high stringency, the PCR was performed with aprecycle of 3 min at 95° C. and 2 min at 60° C., 30 cycles of 20 sec at72° C., 20 sec at 95° C., 20 sec at 60° C. followed by a cycle of 1 minat 72° C. At low stringency, the PCR was performed with a precycle of 5min at 95° C., 35 cycles of 2 min at 40° C., 1 min 30 sec at 72° C., 30sec. at 95° C. followed by a cycle of 4 min at 40° C. and 12 min at 72°C. The oligonucleotides used at high stringency are indicated in theTable below.

PRIMER vgaB Oligo I 5′-AA GTCGAC TGACAATATGAGTGGTGG-3′ (SEQ ID NO: 17)      SalI Oligo II 5′- CTGCAG ATGCCTCAACAGCATCGATATCC-3′ (SEQ ID NO:18)      PstI vatC Oligo III 5′-AT GAATTC GCAAATCAGCAAGG-3′ (SEQ ID NO:19)       EcoRI Oligo IV 5′-TCGTCTC GAGCTC TAGGTCC-3′ (SEQ ID NO: 20)            SacI vgbB Oligo V 5′-CAGCAG TCTAGA TCAGAGTGG-3′ (SEQ ID NO:21)            XbaI Oligo VI 5′-CATAC GGATCC ACCTTTTCC-3′ (SEQ ID NO:22)          BamH1

Example 10 Labelling of DNA Probes

Plasmid DNA was labelled with [α−³²P]dCTP (110 Tbq mmol⁻¹) by the randomprinting technique using the Megaprime DNA labelling system (Amersham).

Example 11 Blotting and Hybridization

Hybond-N+membranes (Amersham) were used for blotting. DNA wastransferred from agarose gels to the membranes by the capillary blottingmethod of Southern Blotting. DNA was denatured and fixed to themembranes according to the protocol described in the handbook user ofHybond-N+membranes.

Prehybridization and hybridization were done at 68° C. in a mixturecontaining 5×SSPE (1×SSPE is 0.3 M NaCl, 30 m tri-sodium citrate),5×Denhardt's solution, 0.5% (w/v) SDS, and 100 μg ml⁻¹ salmon sperm DNA.The membranes containing DNA transferred from agarose gels were treatedwith 10 μg ml⁻¹ radiolabeled DNA probe. Washing was started with twosuccessive immersions in 2×SSPE, 0.1% SDS, at room temperature for 10min, followed by one immersion in 1×SSPE, 0.1% SDS, at 68° C. for 15min, and finally by one immersion in 0.1×SSPE, 0.1% SDS, at 68° C. for15 min. The washed blots treated with the radiolabeled probe wereexposed to Fuji RX film at −70° C.

Example 12 Nucleotides Sequence Determination

For vatC and vgbB, the sequencing reaction was performed by PCRamplification in a final volume of 20 μl using 500 ng of plasmid DNA,5-10 pmoles of primer and 9.5 μl of DyeTerminators premix according toApplied Biosystems protocol. After heating to 94° C. for 2 min., thereaction was cycled as the following: 25 cycles of 30 s at 94° C., 30 sat 55° C., and 4 min at 60° C. (9600 thermal cycler Perkin Elmer).Removal of excess of DyeTerminators were performed using Quick Spincolumns (Boehringer Mannheim). The samples were dried in a vacuumcentrifuge and dissolved with 4 μl of deionized formamide EDTA pH 8.0(5/1). The samples were loaded onto an Applied Biosystems 373A sequencerand run for 12 h on a 4.5% denaturing acrylamide gel.

Primers used for sequencing the following genes:

• vatC 5′-GAAATGGTTGGGAGAAGCATACC-3′ SEQ ID NO: 235′-CAGCAATCGCGCCCGTTTG-3′ SEQ ID NO: 24 5′-AATCGGCAGAATTACAAACG-3′ SEQID NO: 25 5′-CGTTCCCAATTTCCGTGTTACC-3′ SEQ ID NO: 26 • vatB5′-GTTTCTATGCTGATCTGAATC-3′ SEQ ID NO: 27 5′-GTCGTTTGTAATTCTGCCGATT-3′SEQ ID NO: 28 5′-GGTCTAAATGGCGATATATGG-3′ SEQ ID NO: 295′-TTCGAATTCTTTTATCCTACC-3′ SEQ ID NO: 30

For vgaB. DNA was sequenced according to the instructions provided bythe T7SequencinTm kit from Pharmacia Biotech (Uppsala, Sweden),procedures C and D.

Primers used for sequencing the following genes:

• vgaB 5-GCTTGGCAAAAGCAACC-3 (SEQ ID NO: 31) 5-TGAATATAGGATAG-3 (SEQ IDNO: 32) 5-TTGGATCAGGGCC-3 (SEQ ID NO: 33) 5-CAATTAGAAGAACCAC-3 (SEQ IDNO: 34) 5-CAATTGTTCAGCTAGG-3 (SEQ ID NO: 35) 5-GAATTCATTCTATGG-3 (SEQ IDNO: 36) 5-TACACCATTGTTACC-3 (SEQ ID NO: 37) 5-CAAGGAATGATTAAGCC-3 (SEQID NO: 38) 5-GATTCAGATGTTCCC-3 (SEQ ID NO: 39) 5-TCATGGTCGCAATG-3 (SEQID NO: 40) 5-GTTGCTTTCGTAGAAGC-3 (SEQ ID NO: 41) 5-GTTATGTCATCCTC-3 (SEQID NO: 42) 5-GGTTCATCTACGAGC-3 (SEQ ID NO: 43) 5-GGATATCGATGCTG-3 (SEQID NO: 44) 5-GCCAACTCCATTC-3 (SEQ ID NO: 45) 5-CCTAGCTGAACAATTG-3 (SEQID NO: 46) 5-GAAGGTGCCTGATCC-3 (SEQ ID NO: 31) 5-ATACTAGAAATGC-3 (SEQ IDNO: 48)

Example 13 DNA Cloning

A standard protocol was followed for cloning into the vector pOX7, alsonamed pOX300, the 2.4 kb Hindffl-HaeIII fragment of pIP1633 carring vgaB(FIG. 1) and the plasmid pIP1714 carrying vatC and vgbB (FIG. 4),linearized by cleavage with HindIII. The vector DNA (10-20 μg) and theplasmids used in cloning experiments were cleaved with the appropriaterestriction enzymes (30 Units) and purified by GeneClean Kit (Bio 101,La Jolla, Calif.). To avoid religation, the vector cleaved with a singleenzyme was dephosphorylated by 30 min incubation at 37° C. with 5 Unitsof alcaline phosphatase. Ligation was carried out in a total reactionvolume of 10 μl containing 0.1 μg of the vector, 0.1 μg of the plasmid,0.5 mM ATP, 1×T4 DNA ligase buffer and 0.1 Weiss Unit of T4 DNA ligase.After overnight incubation at 16° C., 1 to 2 pl of the ligation mixtureare used for transforming competent E. Coli and the transformants wereselected on solid media containing 100 μg/ml of ampicillin.

Example 14 Susceptibility to Antimicrobial Agents

Susceptibility to antimicrobial agents was determined with a diskdiffusion assay and commercially available disks (Diagnostic Pasteur).Additional disks prepared in our laboratory contained streptogramin A(20 μg) or streptogramin B (40 μg).

NCCLS: Performance standards for antimicrobial disk susceptibility test,1984, Approved standard M2-A3, 4:369-402. ECCLS: Standard forantimicrobial susceptibility testing by diffusion methods, 1985, ECCLSDocument, 5:4-14.

Minimal inhibitory concentrations (MICs) of antibiotics were determinedby serial twofold dilutions of antibiotics in MHA (Ericson H. M. and S.C. Sherris, ActaPathol. Microbiol. Scand., 1971, Suppl. 217:Section B).

Despite the relatively low frequency of detection of SgA^(R)staphylococci (1-10%) (Loncle et al., 1993; Allignet et al., 1996), fourgenes encoding resistance to streptogramin A have been detected andother resistance gene(s) are suspected to be carried by staphylococci.Surprisingly, the present and previous studies (Allignet et al., 1996)indicate that staphylococcal plasmids carrying two genes encodingstreptogramin A resistance by two distinct mechanisms (inactivation byacetyltransferases and increased efflux) are widespread amongstaphylococci (32 of the 48 plasmids investigated).

REFERENCES

The following publications have been cited herein. The entire disclosureof each publication is relied upon and incorporated by reference herein.

Allignet, J., Loncle, V., Mazodier, P. and El Solh, N. (1988) Nucleotidesequence of a staphylococcal plasmid gene, vgb, encoding a hydrolaseinactivating the B components of virginiamycin-like antibiotics. Plasmid20, 271-275.

Allignet, J., Loncle, V. and El Solh, N. (1992) Sequence of astaphylococcal plasmid gene, vga, encoding a putative ATP-bindingprotein involved in resistance to virginiamycin A-like antibiotics. Gene117, 45-51.

Allignet, J., Loncle, V., Simenel, C., Delepierre, M. and El Solh, N.(1993) Sequence of a staphylococcal gene, vat, encoding anacetyltransferase inactivating the A-type compounds ofvirginiamycin-like antibiotics. Gene 130, 91-98.

Allignet, J. and El Solh, N. (1995) Diversity among the Gram-positiveacetyltransferases inactivating streptogramin A and structurally relatedcompounds, and characterization of a new staphylococcal determinant,vatB. Antimicrob. Agents Chemother. 39, 2027-2036.

Allignet, J., Aubert, S., Morvan, A. and El Solh, N. (1996) Distributionof the genes encoding resistance to streptogramin A and relatedcompounds among the staphylococci resistant to these antibiotics.Antimicrob. Agents Chemother. 40, 2523-2528.

Allignet, J. and El Solh, N. (1996) Sequence of a staphylococcal plasmidgene vga B, encoding a putative ATP-binding protein related to vgainvolved in resistance to streptogramin A, 8th International Symposiumon Staphylococci and Staphylococcal Infections, Jun. 23-26, 1996, p.202, 239.

Aumercier, M., Bouhallab, S., Capmau, M. L. and Le Goffic, F. (1992)RP59500: a proposed mechanism for its bactericidal activity. J.Antimicrob. Chemother. 30, 9-14.

Barrasa, M. i., Tercero, J. A., Lacalle, R. A. and Jimenez, A. (1995)The ardl gene from Streptomyces capreolus encodes a polypeptide of theABC-transporters superfamily which confers resistance to theamino-nucleotide antibiotic A201A. Eur. J. Biochem. 228, 562-569.

Blanc, V., Salah-Bey, K., Folcher, M. and Thompson, C. J. (1995)Molecular characterization and transcriptional analysis of a multidrugresistance gene cloned from the pristinamycin-producing organism,Streptomyces pristinaespiralis. Mol. Microbiol. 17, 989-999.

Cocito, C. (1979) Antibiotics of the virginiamycin family, inhibitorswhich contain synergistic components. Microbiol. Rev. 43, 145-198.

Di Giambattista, M., Chinali, G. and Cocito, C. (1989) The molecularbasis of the inhibitory activities of type A and type B synergimycinsand related antibiotics on ribosomes. J. Antimicrob. Chemother. 24,485-507.

Dyke, K. G. H. and Curnock, S. P. (1989) The nucleotide sequence of asmall crypticplasmid found in Staphylococcus aureus and its relationshipto other plasmids. FEMS Microbiol. Lett. 58, 209-216.

El Solh, N., Fouace, J. M., Shalita, Z., Bouanchaud, D. H., Novick, R.P. and Chabbert, Y. A. (1980) Epidemiological and structural studies ofStaphylococcus aureus R plasmids mediating resistance to tobramycin andstreptogramin. Plasmid 4, 117-120.

Entenza, J. M., Drugeon, H., Glauser, M. P. and Moreillon, P. (1995)Treatment of experimental endocarditis due to erythromycin-susceptibleor -resistant methicillin-resistant Staphylococcus aureus with RP59500.Antimicrob. Agents Chemother. 39, 1419-1424.

Fantin, B., Leclercq, R., Merl, Y., Saint-Julien, L., Veyrat, C., Duval,J. and Carbon, C. (1995) Critical influence of resistance tostreptogramin B-type antibiotics on activity of RP59500(quinupristin-dalfopristin) in experimental endocarditis due tohylococcus aureus. Antimicrob. Agents Chemother. 39, 400-405.

Fierro, J. F., Vilches, C., Hardisson, C. and Salas, J. A. (1989)Streptogramins-inactivating activity in three producer streptomycetes.FEMS Microbiol. Lett. 58, 243-246.

Geistlich, M., Losick, R., Turner, J. R. and Rao, R. N. (1992)Characterization of a novel regulatory gene governing the expression ofa polyketide synthase gene in Streptomyces ambofaciens. Mol. Microbiol.6, 2019-2029.

Griswold, M. W., Lomaestro, B. M. and Briceland, L. L. (1996)Quinupristin-dalfopristin (RP59500)—an injectable streptogramincombination. Amer. J. Health-Syst. Pharm. 53, 2045-2053.

Hyde, S. C., Emsley, P., Hartshorn, M. J., Mimmack, M. M., Gileadi, U.,Pearce, S. R., Gallagher, M. P., Gill, D. R., Hubbard, R. E. andHiggins, C. F. (1990) Structural model of ATP-binding proteinsassociated with cystic fibrosis, multidrug resistance and bacterialtransport. Nature 346, 362-365.

Kim, C. H., Otake, N. and Yonehara, H. (1974) Studies on mikamycin Blactonase. I. Degradation of mikamycin B by Streptomyces mitakaensis. J.Antibiot. 27, 903-908.

Kloos, W. E. and Schleifer, K. H. (1986). Genus IV. StaphylococcusRosenbach 1884. 18AL, (Nom. Cons. ( )pin. 17 Jud. Comm. 1958, 153). In:Sneath, P. H. A., Mair, N. S., Sharpe, M. E. and Holt, J. G. (Eds.),Bergey's manual of systematic bacteriology. Williams & Wilkins,Baltimore, Vol. 2, pp. 1013-103.

Kreiswirth, B. N., Lofdahl, S., Bethey, M. J., O'Reilly, M., Shlievert,P. M., Bergdoll, M. S. and Novick, R. P. (1983) The toxic shock exotoxinstructural gene is not detectably transmitted by a prophage. Nature 306,709-712.

Kyte, J. and Doolittle, R. F. (1982) A simple method for displaying thehydropathic character of a protein. J. Mol. Biol. 157, 105-132.

Liassine, N., Allignet, J. Morvan, A., Aubert, S. and El Solh, N. (1997)Multiplicity of the genes and plasmids conferring resistance topristinamycin in Staphylococci selected in an Algerian hospital, Zbl.Bakt. 1212.

Loncle, V., Casetta, A., Buu-Ho, A. and El Solh, N. (1993) Analysis ofpristinamycin-resistant Staphylococcus epidermidis isolates responsiblefor an outbreak in a parisian hospital. Antimicrob. Agents Chemother.37, 2159-2165.

MacLaughlin, J. R., Murray, C. L. and Rabinowitz, C. (1981) Uniquefeatures in the ribosome binding site sequence of the Gram-positiveStaphylococcus aureus §-lactamase gene. J. Biol. Chem. 256, 11283-11291.

Meyer, C., Bierbaum, G., Heidrich, C., Reis, M., SŸling, J.,Iglesias-Wind, M., Kempter, C., Molitor, E. and Sahl, H.-G. (1995)Nucleotide sequence of the lantibiotic Pep5 biosynthetic gene clusterand functional analysis of PepP and PepC: Evidence for a role of PepC inthioether formation. Eur. J. Biochem. 232, 478-489.

Moran, C. P., Jr., Lang, N., LeGrice, S. F. J., Lee, G., Stephens, M.,Sonenshein, A. L., Pero, J. and Losick, R. (1982) Nucleotide sequencesthat signal the initiation of transcription and translation in Bacillussublilis. Mol. Gen. Genet. 186, 339-346.

Olano, C., Rodriguez, A. M., Mndez, C. and Salas, J. A. (1995) A secondABC transporter is involved in oleandomycin resistance and its secretionby Streptomyces antibioticus. Mol. Microbiol. 16,333-343.

Peschke, U., Schmidt, H., Zhang, H.-Z. and Piepersberg, W. (1995)Molecular characterization of the lincomycin-production gene cluster ofStreptomyces lincolnensis 78-11. Mol. Microbiol. 16, 1137-1156.

Rende-Fournier, R., Leclercq, R., Galimand, M., Duval, J. and Courvalin,P. (1993) identification of the satA gene encoding a streptogramin Aacetyltransferase in Enterococcus faecium BM4145. Antimicrob. AgentsChemother. 37, 2119-2125.

Ross, J. I., Eady, E. A., Cove, J. H., Cunliffe, W. J., Baumberg, S. andWootton, J. C. (1990) Inducible erythromycin resistance in staphylococciis encoded by a member of the ATP-binding transport super-gene family.Mol. Microbiol. 4(7), 1207-1214.

Ross, J. I., Eady, E. A., Cove, J. H. and Baumberg, S. (1995)Identification of a chromosomally encoded ABC-transport system withwhich the staphylococcal erythromycin exporter MsrA may interact. Gene153, 93-98.

Ross, J. I., Eady, E. A., Cove, H. H. and Baumberg, S. (1996) Minimalfunctional system required for expression of erythromycin resistance byMSRA in Staphyloocccus aureus RN4220. Gene 183, 143-148.

Rosteck, P. R. J., Reynolds, P. A. and Hershberger, C. L. (1991)Homology between proteins controlling Streptomyces fradiae tylosinresistance and ATP-binding transport. Gene 102, 27-32.

Sanger, F., Nicklen, S. and Coulson, A. R. (1977) DNA sequencing withchain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463-5467.

Schoner, B., Geistlich, M., Rosteck, P. R., Jr., Rao, R. N., Seno, E.,Reynolds, P., Cox, K., Burgett, S. and Hershberger, C. (1992) Sequencesimilarity between macrolide-resistance determinants and ATP-bindingtransport proteins. Gene 115, 93-96.

Southern, E. M. (1975) Detection of specific sequences among DNAfragments separated by gel electrophoresis. J. Mol. Biol. 98, 503-517.

Tinoco, I., Jr., Borer, P. N., Dengler, B., Levine, M. D., Uhlenbeck, O.C., Crothers, D. M. and Gralla, J. (1973) Improved estimation ofsecondary structure in ribonucleic acids. Nature New Biol. 246, 40-41.

Torralba, M. D., Frey, S. E. and Lagging, L. M. (1995) Treatment ofmethicillin-resistant Staphylococcus aureus infection with quinupristindalfopristin. Clin. Infect. Dis. 21, 460-461.

von Heijne, G. (1986) A new method for predicting signal sequencecleavage sites. Nucl. Acids Res. 14, 4683-4690.

Walker, J. E., Saraste, M., Runswick, M. J. and Gay, N. J. (1982)Distantly related sequences in the a- and §-subunits of ATP synthase,myosin, kinases and other ATP-requiring enzymes and a common nucleotidebinding fold. EMBO J. 1, 945-951.

Watson, M. E. E. (1984) Compilation of published signal sequences. Nucl.Acids Res. 12, 5145-5148.

51 1 1656 DNA Staphylococcus sp. 1 atgcttaaaa tcgacatgaa gaatgtaaaaaaatattatg cagataaatt aattttaaat 60 ataaaagaac taaagattta tagtggggataaaataggta ttgtaggtaa gaatggagtt 120 ggcaaaacaa cacttttaaa aataataaaaggactaatag agattgacga aggaaatata 180 attataagtg aaaaaacaac tattaaatatatctctcaat tagaagaacc acatagtaag 240 ataattgatg gaaaatatgc ttcaatatttcaagttgaaa ataagtggaa tgacaatatg 300 agtggtggtg aaaaaactag atttaaactagcagagggat ttcaagatca atgttcttta 360 atgctcgtag atgaacctac aagtaatttagatatcgaag gaatagagtt gataacaaat 420 acttttaaag agtaccgtga tacttttttggtagtatctc atgatagaat ttttttagat 480 caagtttgta caaaaatttt tgaaattgaaaatggatata ttagagaatt catcggtaat 540 tatacaaact atatagagca aaaagaaatgcttctacgaa agcaacaaga agaatacgaa 600 aagtataatt ctaaaagaaa gcaattggagcaagctataa agctaaaaga gaataaggcg 660 caaggaatga ttaagccccc ttcaaaaacaatgggaacat ctgaatctag aatatggaaa 720 atgcaacatg ctactaaaca aaaaaagatgcatagaaata cgaaatcgtt ggaaacacga 780 atagataaat taaatcatgt agaaaaaataaaagagcttc cttctattaa aatggattta 840 cctaatagag agcaatttca tggtcgcaatgtaattagtt taaaaaactt atctataaaa 900 tttaataatc aatttctttg gagagatgcttcatttgtca ttaaaggtgg agaaaaggtt 960 gctataattg gtaacaatgg tgtaggaaaaacaacattgt tgaagctgat tctagaaaaa 1020 gtagaatcag taataatatc accatcagttaaaattggat acgtcagtca aaacttagat 1080 gttctacaat ctcataaatc tatcttagaaaatgttatgt ctacctccat tcaagatgaa 1140 acaatagcaa gaattgttct agcaagattacatttttatc gcaatgatgt tcataaagaa 1200 ataaatgttt tgagtggtgg agaacaaataaaggttgctt ttgccaagct atttgttagc 1260 gattgtaata cattaattct tgatgaaccaacaaactatt tggatatcga tgctgttgag 1320 gcattagaag aattgttaat tacctatgaaggtgttgtgt tatttgcttc ccatgataaa 1380 aaatttatac aaaacctagc tgaacaattgttaataatag aaaataataa agtgaaaaaa 1440 ttcgaaggaa catatataga atatttaaaaattaaagata aaccaaaatt aaatacaaat 1500 gaaaaagaac tcaaagaaaa aaagatgatactagaaatgc aaatttcatc attattaagt 1560 aaaatctcaa tggaagaaaa tgaagaaaaaaacaaagaat tagatgaaaa gtacaaattg 1620 aaattaaaag aattgaaaag cctaaataaaaatatt 1656 2 636 DNA Staphylococcus sp. 2 atgaaatggc aaaatcagcaaggccccaat ccagaagaaa tataccctat agaaggtaat 60 aaacatgttc aatttattaaaccatctata acaaagccca atattttagt tggggaatat 120 tcatattacg atagtaaagatggtgaatct tttgaaagcc aagttcttta tcactatgaa 180 ttgattgggg ataaactaatattagggaag ttttgttcta ttggacccgg aacgacattt 240 ataatgaatg gggctaatcatcgtatggat ggttcaacat ttccattcaa tcttttcgga 300 aatggttggg agaagcatacccctacattg gaagaccttc cttataaggg taacacggaa 360 attgggaacg atgtttggattggacgagat gtgacaatta tgcccggtgt aaaaatagga 420 aacggggcta ttattgcagcaaaatcggtt gtgacaaaga acgttgatcc ttattcagtt 480 gttggcggta atccttcacgattaattaag ataaggtttt ccaaggaaaa aatcgcagca 540 ttactaaaag taaggtggtgggacctagag atagagacga taaatgaaaa tattgattgc 600 atcctgaatg gtgatataaaaaaggttaaa agaagt 636 3 885 DNA Staphylococcus sp. 3 atgaatttttatttagagga gtttaacttg tctattcccg attcaggtcc atacggtata 60 acttcatcagaagacggaaa ggtatggttc acacaacata aggcaaacaa aatcagcagt 120 ctagatcagagtggtaggat aaaagaattc gaagttccta cccctgatgc taaagtgatg 180 tgtttaattgtatcttcact tggagacata tggtttacag agaatggtgc aaataaaatc 240 ggaaagctctcaaaaaaagg tggctttaca gaatatccat tgccacagcc ggattctggt 300 ccttacggaataacggaagg tctaaatggc gatatatggt ttacccaatt gaatggagat 360 cgtataggaaagttgacagc tgatgggact atttatgaat atgatttgcc aaataaggga 420 tcttatcctgcttttattac tttaggttcg gataacgcac tttggttcac ggagaaccaa 480 aataattctattggaaggat tacaaataca gggaaattag aagaatatcc tctaccaaca 540 aatgcagcggctccagtggg tatcactagt ggtaacgatg gtgcactctg gtttgtcgaa 600 attatgggcaacaaaatagg tcgaatcact acaactggtg agattagcga atatgatatt 660 ccaactccaaacgcacgtcc acacgctata accgcgggga aaaatagcga aatatggttt 720 actgaatggggggcaaatca aatcggcaga attacaaacg acaaaacaat tcaagaatat 780 caacttcaaacagaaaatgc ggaacctcat ggtattacct ttggaaaaga tggatccgta 840 tggtttgcattaaaatgtaa aattgggaag ctgaatttga acgaa 885 4 552 PRT Staphylococcus sp.4 Met Leu Lys Ile Asp Met Lys Asn Val Lys Lys Tyr Tyr Ala Asp Lys 1 5 1015 Leu Ile Leu Asn Ile Lys Glu Leu Lys Ile Tyr Ser Gly Asp Lys Ile 20 2530 Gly Ile Val Gly Lys Asn Gly Val Gly Lys Thr Thr Leu Leu Lys Ile 35 4045 Ile Lys Gly Leu Ile Glu Ile Asp Glu Gly Asn Ile Ile Ile Ser Glu 50 5560 Lys Thr Thr Ile Lys Tyr Ile Ser Gln Leu Glu Glu Pro His Ser Lys 65 7075 80 Ile Ile Asp Gly Lys Tyr Ala Ser Ile Phe Gln Val Glu Asn Lys Trp 8590 95 Asn Asp Asn Met Ser Gly Gly Glu Lys Thr Arg Phe Lys Leu Ala Glu100 105 110 Gly Phe Gln Asp Gln Cys Ser Leu Met Leu Val Asp Glu Pro ThrSer 115 120 125 Asn Leu Asp Ile Glu Gly Ile Glu Leu Ile Thr Asn Thr PheLys Glu 130 135 140 Tyr Arg Asp Thr Phe Leu Val Val Ser His Asp Arg IlePhe Leu Asp 145 150 155 160 Gln Val Cys Thr Lys Ile Phe Glu Ile Glu AsnGly Tyr Ile Arg Glu 165 170 175 Phe Ile Gly Asn Tyr Thr Asn Tyr Ile GluGln Lys Glu Met Leu Leu 180 185 190 Arg Lys Gln Gln Glu Glu Tyr Glu LysTyr Asn Ser Lys Arg Lys Gln 195 200 205 Leu Glu Gln Ala Ile Lys Leu LysGlu Asn Lys Ala Gln Gly Met Ile 210 215 220 Lys Pro Pro Ser Lys Thr MetGly Thr Ser Glu Ser Arg Ile Trp Lys 225 230 235 240 Met Gln His Ala ThrLys Gln Lys Lys Met His Arg Asn Thr Lys Ser 245 250 255 Leu Glu Thr ArgIle Asp Lys Leu Asn His Val Glu Lys Ile Lys Glu 260 265 270 Leu Pro SerIle Lys Met Asp Leu Pro Asn Arg Glu Gln Phe His Gly 275 280 285 Arg AsnVal Ile Ser Leu Lys Asn Leu Ser Ile Lys Phe Asn Asn Gln 290 295 300 PheLeu Trp Arg Asp Ala Ser Phe Val Ile Lys Gly Gly Glu Lys Val 305 310 315320 Ala Ile Ile Gly Asn Asn Gly Val Gly Lys Thr Thr Leu Leu Lys Leu 325330 335 Ile Leu Glu Lys Val Glu Ser Val Ile Ile Ser Pro Ser Val Lys Ile340 345 350 Gly Tyr Val Ser Gln Asn Leu Asp Val Leu Gln Ser His Lys SerIle 355 360 365 Leu Glu Asn Val Met Ser Thr Ser Ile Gln Asp Glu Thr IleAla Arg 370 375 380 Ile Val Leu Ala Arg Leu His Phe Tyr Arg Asn Asp ValHis Lys Glu 385 390 395 400 Ile Asn Val Leu Ser Gly Gly Glu Gln Ile LysVal Ala Phe Ala Lys 405 410 415 Leu Phe Val Ser Asp Cys Asn Thr Leu IleLeu Asp Glu Pro Thr Asn 420 425 430 Tyr Leu Asp Ile Asp Ala Val Glu AlaLeu Glu Glu Leu Leu Ile Thr 435 440 445 Tyr Glu Gly Val Val Leu Phe AlaSer His Asp Lys Lys Phe Ile Gln 450 455 460 Asn Leu Ala Glu Gln Leu LeuIle Ile Glu Asn Asn Lys Val Lys Lys 465 470 475 480 Phe Glu Gly Thr TyrIle Glu Tyr Leu Lys Ile Lys Asp Lys Pro Lys 485 490 495 Leu Asn Thr AsnGlu Lys Glu Leu Lys Glu Lys Lys Met Ile Leu Glu 500 505 510 Met Gln IleSer Ser Leu Leu Ser Lys Ile Ser Met Glu Glu Asn Glu 515 520 525 Glu LysAsn Lys Glu Leu Asp Glu Lys Tyr Lys Leu Lys Leu Lys Glu 530 535 540 LeuLys Ser Leu Asn Lys Asn Ile 545 550 5 212 PRT Staphylococcus sp. 5 MetLys Trp Gln Asn Gln Gln Gly Pro Asn Pro Glu Glu Ile Tyr Pro 1 5 10 15Ile Glu Gly Asn Lys His Val Gln Phe Ile Lys Pro Ser Ile Thr Lys 20 25 30Pro Asn Ile Leu Val Gly Glu Tyr Ser Tyr Tyr Asp Ser Lys Asp Gly 35 40 45Glu Ser Phe Glu Ser Gln Val Leu Tyr His Tyr Glu Leu Ile Gly Asp 50 55 60Lys Leu Ile Leu Gly Lys Phe Cys Ser Ile Gly Pro Gly Thr Thr Phe 65 70 7580 Ile Met Asn Gly Ala Asn His Arg Met Asp Gly Ser Thr Phe Pro Phe 85 9095 Asn Leu Phe Gly Asn Gly Trp Glu Lys His Thr Pro Thr Leu Glu Asp 100105 110 Leu Pro Tyr Lys Gly Asn Thr Glu Ile Gly Asn Asp Val Trp Ile Gly115 120 125 Arg Asp Val Thr Ile Met Pro Gly Val Lys Ile Gly Asn Gly AlaIle 130 135 140 Ile Ala Ala Lys Ser Val Val Thr Lys Asn Val Asp Pro TyrSer Val 145 150 155 160 Val Gly Gly Asn Pro Ser Arg Leu Ile Lys Ile ArgPhe Ser Lys Glu 165 170 175 Lys Ile Ala Ala Leu Leu Lys Val Arg Trp TrpAsp Leu Glu Ile Glu 180 185 190 Thr Ile Asn Glu Asn Ile Asp Cys Ile LeuAsn Gly Asp Ile Lys Lys 195 200 205 Val Lys Arg Ser 210 6 294 PRTStaphylococcus sp. 6 Met Asn Phe Tyr Leu Glu Glu Phe Asn Leu Ser Ile ProAsp Ser Gly 1 5 10 15 Pro Tyr Gly Ile Thr Ser Ser Glu Asp Gly Lys ValTrp Phe Thr Gln 20 25 30 His Lys Ala Asn Lys Ile Ser Ser Leu Asp Gln SerGly Arg Ile Lys 35 40 45 Glu Phe Glu Val Pro Thr Pro Asp Ala Lys Val MetCys Leu Ile Val 50 55 60 Ser Ser Leu Gly Asp Ile Trp Phe Thr Glu Asn GlyAla Asn Lys Ile 65 70 75 80 Gly Lys Leu Ser Lys Lys Gly Gly Phe Thr GluTyr Pro Leu Pro Gln 85 90 95 Pro Asp Ser Gly Pro Gly Ile Thr Glu Gly LeuAsn Gly Asp Ile Trp 100 105 110 Phe Thr Gln Leu Asn Gly Asp Arg Ile GlyLys Leu Thr Ala Asp Gly 115 120 125 Thr Ile Tyr Glu Tyr Asp Leu Pro AsnLys Gly Ser Tyr Pro Ala Phe 130 135 140 Ile Thr Leu Gly Ser Asp Asn AlaLeu Trp Phe Thr Glu Asn Gln Asn 145 150 155 160 Asn Ser Ile Gly Arg IleThr Asn Thr Gly Lys Leu Glu Glu Tyr Pro 165 170 175 Leu Pro Thr Asn AlaAla Ala Pro Val Gly Ile Thr Ser Gly Asn Asp 180 185 190 Gly Ala Leu TrpPhe Val Glu Ile Met Gly Asn Lys Ile Gly Arg Ile 195 200 205 Thr Thr ThrGly Glu Ile Ser Glu Tyr Asp Ile Pro Thr Pro Asn Ala 210 215 220 Arg ProHis Ala Ile Thr Ala Gly Lys Asn Ser Glu Ile Trp Phe Thr 225 230 235 240Glu Trp Gly Ala Asn Gln Ile Gly Arg Ile Thr Asn Asp Lys Thr Ile 245 250255 Gln Glu Tyr Gln Leu Gln Thr Glu Asn Ala Glu Pro His Gly Ile Thr 260265 270 Phe Gly Lys Asp Gly Ser Val Trp Phe Ala Leu Lys Cys Lys Ile Gly275 280 285 Lys Leu Asn Leu Asn Glu 290 7 7 PRT Staphylococcus sp. 7 LysSer Ile Leu Glu Asn Val 1 5 8 10 PRT Staphylococcus sp. MOD_RES (3) Thror Ser 8 Asn Tyr Xaa Asn Tyr Xaa Glu Gln Lys Glu 1 5 10 9 9 PRTStaphylococcus sp. 9 Ile Met Asn Gly Ala Asn His Arg Met 1 5 10 7 PRTStaphylococcus sp. 10 Gly Asn Asp Val Trp Ile Gly 1 5 11 21 DNAStaphylococcus sp. 11 aarwsyatyt tagaaaatgt t 21 12 30 DNAStaphylococcus sp. 12 aattataswa actatrtwga gcaaaaagaa 30 13 27 DNAStaphylococcus sp. 13 atwatgaatg gkgcwaayca tmgdatg 27 14 21 DNAStaphylococcus sp. 14 ggraaygatg tdtggatwgg w 21 15 2411 DNAStaphylococcus sp. CDS (700..2355, 2388..2411) 15 aagctttaat taagttagcagaagattatg gagtaatact aagaacaagt gatagtagta 60 ctaaagaaca agcaaaagaacaagctaaag atgatataat tgacttatta aaagagcaat 120 tagaatatga aaaagatcgaaatgaaaaac tatcaaaact taacgataat ttattggaac 180 agttagataa aaatcaaacattattagatc agcaacaaag attaagtctt aatgatcaaa 240 atagtatcaa aatgttagaatcagaattag aagaaaaaaa gaagaaaaag aagaaaaaga 300 aactaagtgg tatcatgtattccagagaaa aaaataatta tatattaaaa tgagatacaa 360 caaatgaatt agtttgtttcaataggaatt tggtaaaacc catgtacata taacttttaa 420 tttagtataa ttaaataaacaaagaaatcg aaagggtgaa atattaataa aatgatcaaa 480 taatccgtca ctaaaaagaaaattaaatat attggaaaga ttttacctaa tatatttatg 540 tctatttatt atgattggatagtttgttta tttgttatat ttcacttata taaactatcc 600 tctattttaa aaaaaggaggatttttttat gcttttgttt atttgttata tttcacttat 660 ataaactatc ctctattttaaaaaaaggag gattttttt atg ctt aaa atc gac 714 Met Leu Lys Ile Asp 1 5 atgaag aat gta aaa aaa tat tat gca gat aaa tta att tta aat ata 762 Met LysAsn Val Lys Lys Tyr Tyr Ala Asp Lys Leu Ile Leu Asn Ile 10 15 20 aaa gaacta aag att tat agt ggg gat aaa ata ggt att gta ggt aag 810 Lys Glu LeuLys Ile Tyr Ser Gly Asp Lys Ile Gly Ile Val Gly Lys 25 30 35 aat gga gttggc aaa aca aca ctt tta aaa ata ata aaa gga cta ata 858 Asn Gly Val GlyLys Thr Thr Leu Leu Lys Ile Ile Lys Gly Leu Ile 40 45 50 gag att gac gaagga aat ata att ata agt gaa aaa aca act att aaa 906 Glu Ile Asp Glu GlyAsn Ile Ile Ile Ser Glu Lys Thr Thr Ile Lys 55 60 65 tat atc tct caa ttagaa gaa cca cat agt aag ata att gat gga aaa 954 Tyr Ile Ser Gln Leu GluGlu Pro His Ser Lys Ile Ile Asp Gly Lys 70 75 80 85 tat gct tca ata tttcaa gtt gaa aat aag tgg aat gac aat atg agt 1002 Tyr Ala Ser Ile Phe GlnVal Glu Asn Lys Trp Asn Asp Asn Met Ser 90 95 100 ggt ggt gaa aaa actaga ttt aaa cta gca gag gga ttt caa gat caa 1050 Gly Gly Glu Lys Thr ArgPhe Lys Leu Ala Glu Gly Phe Gln Asp Gln 105 110 115 tgt tct tta atg ctcgta gat gaa cct aca agt aat tta gat atc gaa 1098 Cys Ser Leu Met Leu ValAsp Glu Pro Thr Ser Asn Leu Asp Ile Glu 120 125 130 gga ata gag ttg ataaca aat act ttt aaa gag tac cgt gat act ttt 1146 Gly Ile Glu Leu Ile ThrAsn Thr Phe Lys Glu Tyr Arg Asp Thr Phe 135 140 145 ttg gta gta tct catgat aga att ttt tta gat caa gtt tgt aca aaa 1194 Leu Val Val Ser His AspArg Ile Phe Leu Asp Gln Val Cys Thr Lys 150 155 160 165 att ttt gaa attgaa aat gga tat att aga gaa ttc atc ggt aat tat 1242 Ile Phe Glu Ile GluAsn Gly Tyr Ile Arg Glu Phe Ile Gly Asn Tyr 170 175 180 aca aac tat atagag caa aaa gaa atg ctt cta cga aag caa caa gaa 1290 Thr Asn Tyr Ile GluGln Lys Glu Met Leu Leu Arg Lys Gln Gln Glu 185 190 195 gaa tac gaa aagtat aat tct aaa aga aag caa ttg gag caa gct ata 1338 Glu Tyr Glu Lys TyrAsn Ser Lys Arg Lys Gln Leu Glu Gln Ala Ile 200 205 210 aag cta aaa gagaat aag gcg caa gga atg att aag ccc cct tca aaa 1386 Lys Leu Lys Glu AsnLys Ala Gln Gly Met Ile Lys Pro Pro Ser Lys 215 220 225 aca atg gga acatct gaa tct aga ata tgg aaa atg caa cat gct act 1434 Thr Met Gly Thr SerGlu Ser Arg Ile Trp Lys Met Gln His Ala Thr 230 235 240 245 aaa caa aaaaag atg cat aga aat acg aaa tcg ttg gaa aca cga ata 1482 Lys Gln Lys LysMet His Arg Asn Thr Lys Ser Leu Glu Thr Arg Ile 250 255 260 gat aaa ttaaat cat gta gaa aaa ata aaa gag ctt cct tct att aaa 1530 Asp Lys Leu AsnHis Val Glu Lys Ile Lys Glu Leu Pro Ser Ile Lys 265 270 275 atg gat ttacct aat aga gag caa ttt cat ggt cgc aat gta att agt 1578 Met Asp Leu ProAsn Arg Glu Gln Phe His Gly Arg Asn Val Ile Ser 280 285 290 tta aaa aactta tct ata aaa ttt aat aat caa ttt ctt tgg aga gat 1626 Leu Lys Asn LeuSer Ile Lys Phe Asn Asn Gln Phe Leu Trp Arg Asp 295 300 305 gct tca tttgtc att aaa ggt gga gaa aag gtt gct ata att ggt aac 1674 Ala Ser Phe ValIle Lys Gly Gly Glu Lys Val Ala Ile Ile Gly Asn 310 315 320 325 aat ggtgta gga aaa aca aca ttg ttg aag ctg att cta gaa aaa gta 1722 Asn Gly ValGly Lys Thr Thr Leu Leu Lys Leu Ile Leu Glu Lys Val 330 335 340 gaa tcagta ata ata tca cca tca gtt aaa att gga tac gtc agt caa 1770 Glu Ser ValIle Ile Ser Pro Ser Val Lys Ile Gly Tyr Val Ser Gln 345 350 355 aac ttagat gtt cta caa tct cat aaa tct atc tta gaa aat gtt atg 1818 Asn Leu AspVal Leu Gln Ser His Lys Ser Ile Leu Glu Asn Val Met 360 365 370 tct acctcc att caa gat gaa aca ata gca aga att gtt cta gca aga 1866 Ser Thr SerIle Gln Asp Glu Thr Ile Ala Arg Ile Val Leu Ala Arg 375 380 385 tta catttt tat cgc aat gat gtt cat aaa gaa ata aat gtt ttg agt 1914 Leu His PheTyr Arg Asn Asp Val His Lys Glu Ile Asn Val Leu Ser 390 395 400 405 ggtgga gaa caa ata aag gtt gct ttt gcc aag cta ttt gtt agc gat 1962 Gly GlyGlu Gln Ile Lys Val Ala Phe Ala Lys Leu Phe Val Ser Asp 410 415 420 tgtaat aca tta att ctt gat gaa cca aca aac tat ttg gat atc gat 2010 Cys AsnThr Leu Ile Leu Asp Glu Pro Thr Asn Tyr Leu Asp Ile Asp 425 430 435 gctgtt gag gca tta gaa gaa ttg tta att acc tat gaa ggt gtt gtg 2058 Ala ValGlu Ala Leu Glu Glu Leu Leu Ile Thr Tyr Glu Gly Val Val 440 445 450 ttattt gct tcc cat gat aaa aaa ttt ata caa aac cta gct gaa caa 2106 Leu PheAla Ser His Asp Lys Lys Phe Ile Gln Asn Leu Ala Glu Gln 455 460 465 ttgtta ata ata gaa aat aat aaa gtg aaa aaa ttc gaa gga aca tat 2154 Leu LeuIle Ile Glu Asn Asn Lys Val Lys Lys Phe Glu Gly Thr Tyr 470 475 480 485ata gaa tat tta aaa att aaa gat aaa cca aaa tta aat aca aat gaa 2202 IleGlu Tyr Leu Lys Ile Lys Asp Lys Pro Lys Leu Asn Thr Asn Glu 490 495 500aaa gaa ctc aaa gaa aaa aag atg ata cta gaa atg caa att tca tca 2250 LysGlu Leu Lys Glu Lys Lys Met Ile Leu Glu Met Gln Ile Ser Ser 505 510 515tta tta agt aaa atc tca atg gaa gaa aat gaa gaa aaa aac aaa gaa 2298 LeuLeu Ser Lys Ile Ser Met Glu Glu Asn Glu Glu Lys Asn Lys Glu 520 525 530tta gat gaa aag tac aaa ttg aaa tta aaa gaa ttg aaa agc cta aat 2346 LeuAsp Glu Lys Tyr Lys Leu Lys Leu Lys Glu Leu Lys Ser Leu Asn 535 540 545aaa aat att taaaataaat tatattaata ggaggtttaa aa atg aaa tat ggc 2399 LysAsn Ile Met Lys Tyr Gly 550 555 cct gat cca aat 2411 Pro Asp Pro Asn 56016 1607 DNA Staphylococcus sp. CDS (39..923, 947..1582) 16 aggagtttttgcgttcaaaa taattgggag gaatgtaa atg aat ttt tat tta 53 Met Asn Phe TyrLeu 1 5 gag gag ttt aac ttg tct att ccc gat tca ggt cca tac ggt ata act101 Glu Glu Phe Asn Leu Ser Ile Pro Asp Ser Gly Pro Tyr Gly Ile Thr 1015 20 tca tca gaa gac gga aag gta tgg ttc aca caa cat aag gca aac aaa149 Ser Ser Glu Asp Gly Lys Val Trp Phe Thr Gln His Lys Ala Asn Lys 2530 35 atc agc agt cta gat cag agt ggt agg ata aaa gaa ttc gaa gtt cct197 Ile Ser Ser Leu Asp Gln Ser Gly Arg Ile Lys Glu Phe Glu Val Pro 4045 50 acc cct gat gct aaa gtg atg tgt tta att gta tct tca ctt gga gac245 Thr Pro Asp Ala Lys Val Met Cys Leu Ile Val Ser Ser Leu Gly Asp 5560 65 ata tgg ttt aca gag aat ggt gca aat aaa atc gga aag ctc tca aaa293 Ile Trp Phe Thr Glu Asn Gly Ala Asn Lys Ile Gly Lys Leu Ser Lys 7075 80 85 aaa ggt ggc ttt aca gaa tat cca ttg cca cag ccg gat tct ggt cct341 Lys Gly Gly Phe Thr Glu Tyr Pro Leu Pro Gln Pro Asp Ser Gly Pro 9095 100 tac gga ata acg gaa ggt cta aat ggc gat ata tgg ttt acc caa ttg389 Tyr Gly Ile Thr Glu Gly Leu Asn Gly Asp Ile Trp Phe Thr Gln Leu 105110 115 aat gga gat cgt ata gga aag ttg aca gct gat ggg act att tat gaa437 Asn Gly Asp Arg Ile Gly Lys Leu Thr Ala Asp Gly Thr Ile Tyr Glu 120125 130 tat gat ttg cca aat aag gga tct tat cct gct ttt att act tta ggt485 Tyr Asp Leu Pro Asn Lys Gly Ser Tyr Pro Ala Phe Ile Thr Leu Gly 135140 145 tcg gat aac gca ctt tgg ttc acg gag aac caa aat aat tct att gga533 Ser Asp Asn Ala Leu Trp Phe Thr Glu Asn Gln Asn Asn Ser Ile Gly 150155 160 165 agg att aca aat aca ggg aaa tta gaa gaa tat cct cta cca acaaat 581 Arg Ile Thr Asn Thr Gly Lys Leu Glu Glu Tyr Pro Leu Pro Thr Asn170 175 180 gca gcg gct cca gtg ggt atc act agt ggt aac gat ggt gca ctctgg 629 Ala Ala Ala Pro Val Gly Ile Thr Ser Gly Asn Asp Gly Ala Leu Trp185 190 195 ttt gtc gaa att atg ggc aac aaa ata ggt cga atc act aca actggt 677 Phe Val Glu Ile Met Gly Asn Lys Ile Gly Arg Ile Thr Thr Thr Gly200 205 210 gag att agc gaa tat gat att cca act cca aac gca cgt cca cacgct 725 Glu Ile Ser Glu Tyr Asp Ile Pro Thr Pro Asn Ala Arg Pro His Ala215 220 225 ata acc gcg ggg aaa aat agc gaa ata tgg ttt act gaa tgg ggggca 773 Ile Thr Ala Gly Lys Asn Ser Glu Ile Trp Phe Thr Glu Trp Gly Ala230 235 240 245 aat caa atc ggc aga att aca aac gac aaa aca att caa gaatat caa 821 Asn Gln Ile Gly Arg Ile Thr Asn Asp Lys Thr Ile Gln Glu TyrGln 250 255 260 ctt caa aca gaa aat gcg gaa cct cat ggt att acc ttt ggaaaa gat 869 Leu Gln Thr Glu Asn Ala Glu Pro His Gly Ile Thr Phe Gly LysAsp 265 270 275 gga tcc gta tgg ttt gca tta aaa tgt aaa att ggg aag ctgaat ttg 917 Gly Ser Val Trp Phe Ala Leu Lys Cys Lys Ile Gly Lys Leu AsnLeu 280 285 290 aac gaa tgagatggga gtgagcaata ttt atg aaa tgg caa aatcag caa 967 Asn Glu Met Lys Trp Gln Asn Gln Gln 295 300 ggc ccc aat ccagaa gaa ata tac cct ata gaa ggt aat aaa cat gtt 1015 Gly Pro Asn Pro GluGlu Ile Tyr Pro Ile Glu Gly Asn Lys His Val 305 310 315 caa ttt att aaacca tct ata aca aag ccc aat att tta gtt ggg gaa 1063 Gln Phe Ile Lys ProSer Ile Thr Lys Pro Asn Ile Leu Val Gly Glu 320 325 330 tat tca tat tacgat agt aaa gat ggt gaa tct ttt gaa agc caa gtt 1111 Tyr Ser Tyr Tyr AspSer Lys Asp Gly Glu Ser Phe Glu Ser Gln Val 335 340 345 350 ctt tat cactat gaa ttg att ggg gat aaa cta ata tta ggg aag ttt 1159 Leu Tyr His TyrGlu Leu Ile Gly Asp Lys Leu Ile Leu Gly Lys Phe 355 360 365 tgt tct attgga ccc gga acg aca ttt ata atg aat ggg gct aat cat 1207 Cys Ser Ile GlyPro Gly Thr Thr Phe Ile Met Asn Gly Ala Asn His 370 375 380 cgt atg gatggt tca aca ttt cca ttc aat ctt ttc gga aat ggt tgg 1255 Arg Met Asp GlySer Thr Phe Pro Phe Asn Leu Phe Gly Asn Gly Trp 385 390 395 gag aag catacc cct aca ttg gaa gac ctt cct tat aag ggt aac acg 1303 Glu Lys His ThrPro Thr Leu Glu Asp Leu Pro Tyr Lys Gly Asn Thr 400 405 410 gaa att gggaac gat gtt tgg att gga cga gat gtg aca att atg ccc 1351 Glu Ile Gly AsnAsp Val Trp Ile Gly Arg Asp Val Thr Ile Met Pro 415 420 425 430 ggt gtaaaa ata gga aac ggg gct att att gca gca aaa tcg gtt gtg 1399 Gly Val LysIle Gly Asn Gly Ala Ile Ile Ala Ala Lys Ser Val Val 435 440 445 aca aagaac gtt gat cct tat tca gtt gtt ggc ggt aat cct tca cga 1447 Thr Lys AsnVal Asp Pro Tyr Ser Val Val Gly Gly Asn Pro Ser Arg 450 455 460 tta attaag ata agg ttt tcc aag gaa aaa atc gca gca tta cta aaa 1495 Leu Ile LysIle Arg Phe Ser Lys Glu Lys Ile Ala Ala Leu Leu Lys 465 470 475 gta aggtgg tgg gac cta gag ata gag acg ata aat gaa aat att gat 1543 Val Arg TrpTrp Asp Leu Glu Ile Glu Thr Ile Asn Glu Asn Ile Asp 480 485 490 tgc atcctg aat ggt gat ata aaa aag gtt aaa aga agt tagaaaacga 1592 Cys Ile LeuAsn Gly Asp Ile Lys Lys Val Lys Arg Ser 495 500 505 attttgttta ggtta1607 17 26 DNA Artificial Sequence Description of Artificial SequencePrimer 17 aagtcgactg acaatatgag tggtgg 26 18 29 DNA Artificial SequenceDescription of Artificial Sequence Primer 18 ctgcagatgc ctcaacagcatcgatatcc 29 19 22 DNA Artificial Sequence Description of ArtificialSequence Primer 19 atgaattcgc aaatcagcaa gg 22 20 20 DNA ArtificialSequence Description of Artificial Sequence Primer 20 tcgtctcgagctctaggtcc 20 21 21 DNA Artificial Sequence Description of ArtificialSequence Primer 21 cagcagtcta gatcagagtg g 21 22 20 DNA ArtificialSequence Description of Artificial Sequence Primer 22 catacggatccaccttttcc 20 23 23 DNA Artificial Sequence Description of ArtificialSequence Primer 23 gaaatggttg ggagaagcat acc 23 24 19 DNA ArtificialSequence Description of Artificial Sequence Primer 24 cagcaatcgcgcccgtttg 19 25 20 DNA Artificial Sequence Description of ArtificialSequence Primer 25 aatcggcaga attacaaacg 20 26 22 DNA ArtificialSequence Description of Artificial Sequence Primer 26 cgttcccaatttccgtgtta cc 22 27 21 DNA Artificial Sequence Description of ArtificialSequence Primer 27 gtttctatgc tgatctgaat c 21 28 22 DNA ArtificialSequence Description of Artificial Sequence Primer 28 gtcgtttgtaattctgccga tt 22 29 21 DNA Artificial Sequence Description of ArtificialSequence Primer 29 ggtctaaatg gcgatatatg g 21 30 21 DNA ArtificialSequence Description of Artificial Sequence Primer 30 ttcgaattcttttatcctac c 21 31 17 DNA Artificial Sequence Description of ArtificialSequence Primer 31 gcttggcaaa agcaacc 17 32 14 DNA Artificial SequenceDescription of Artificial Sequence Primer 32 tgaatatagg atag 14 33 13DNA Artificial Sequence Description of Artificial Sequence Primer 33ttggatcagg gcc 13 34 16 DNA Artificial Sequence Description ofArtificial Sequence Primer 34 caattagaag aaccac 16 35 16 DNA ArtificialSequence Description of Artificial Sequence Primer 35 caattgttca gctagg16 36 15 DNA Artificial Sequence Description of Artificial SequencePrimer 36 gaattcattc tatgg 15 37 15 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 37 tacaccattg ttacc 15 38 17 DNAArtificial Sequence Description of Artificial Sequence Primer 38caaggaatga ttaagcc 17 39 15 DNA Artificial Sequence Description ofArtificial Sequence Primer 39 gattcagatg ttccc 15 40 14 DNA ArtificialSequence Description of Artificial Sequence Primer 40 tcatggtcgc aatg 1441 17 DNA Artificial Sequence Description of Artificial Sequence Primer41 gttgctttcg tagaagc 17 42 14 DNA Artificial Sequence Description ofArtificial Sequence Primer 42 gttatgtcat cctc 14 43 15 DNA ArtificialSequence Description of Artificial Sequence Primer 43 ggttcatcta cgagc15 44 14 DNA Artificial Sequence Description of Artificial SequencePrimer 44 ggatatcgat gctg 14 45 13 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 45 gccaactcca ttc 13 46 16 DNA ArtificialSequence Description of Artificial Sequence Primer 46 cctagctgaa caattg16 47 15 DNA Artificial Sequence Description of Artificial SequencePrimer 47 gaaggtgcct gatcc 15 48 13 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 48 atactagaaa tgc 13 49 522 PRTStaphylococcus sp. 49 Met Lys Ile Met Leu Glu Gly Leu Asn Ile Lys HisTyr Val Gln Asp 1 5 10 15 Arg Leu Leu Leu Asn Ile Asn Arg Leu Lys IleTyr Gln Asn Asp Arg 20 25 30 Ile Gly Leu Ile Gly Lys Asn Gly Ser Gly LysThr Thr Leu Leu His 35 40 45 Ile Leu Tyr Lys Lys Ile Val Pro Glu Glu GlyIle Val Lys Gln Phe 50 55 60 Ser His Cys Glu Leu Ile Pro Gln Leu Lys LeuIle Glu Ser Thr Lys 65 70 75 80 Ser Gly Gly Glu Val Thr Arg Asn Tyr IleArg Gln Ala Leu Asp Lys 85 90 95 Asn Pro Glu Leu Leu Leu Ala Asp Glu ProThr Thr Asn Leu Asp Asn 100 105 110 Asn Tyr Ile Glu Lys Leu Glu Gln AspLeu Lys Asn Trp His Gly Ala 115 120 125 Phe Ile Ile Val Ser His Asp ArgAla Phe Leu Asp Asn Leu Cys Thr 130 135 140 Thr Ile Trp Glu Ile Asp GluGly Arg Ile Thr Glu Tyr Lys Gly Asn 145 150 155 160 Tyr Ser Asn Tyr ValGlu Gln Lys Glu Leu Glu Arg His Arg Glu Glu 165 170 175 Leu Glu Tyr GluLys Tyr Glu Lys Glu Lys Lys Arg Leu Glu Lys Ala 180 185 190 Ile Asn IleLys Glu Gln Lys Ala Gln Arg Ala Thr Lys Lys Pro Lys 195 200 205 Asn LeuSer Leu Ser Glu Gly Lys Ile Lys Gly Ala Lys Pro Tyr Phe 210 215 220 AlaGly Lys Gln Lys Lys Leu Arg Lys Thr Val Lys Ser Leu Glu Thr 225 230 235240 Arg Leu Glu Lys Leu Glu Ser Val Glu Lys Arg Asn Glu Leu Pro Pro 245250 255 Leu Lys Met Asp Leu Val Asn Leu Glu Ser Val Lys Asn Arg Thr Ile260 265 270 Ile Arg Gly Glu Asp Val Ser Gly Thr Ile Glu Gly Arg Val LeuTrp 275 280 285 Lys Ala Lys Ser Phe Ser Ile Arg Gly Gly Asp Lys Met AlaIle Ile 290 295 300 Gly Ser Asn Gly Thr Gly Lys Thr Thr Phe Ile Lys LysIle Val His 305 310 315 320 Gly Asn Pro Gly Ile Ser Leu Ser Pro Ser ValLys Ile Gly Tyr Phe 325 330 335 Ser Gln Lys Ile Asp Thr Leu Glu Leu AspLys Ser Ile Leu Glu Asn 340 345 350 Val Gln Ser Ser Ser Gln Gln Asn GluThr Leu Ile Arg Thr Ile Leu 355 360 365 Ala Arg Met His Phe Phe Arg AspAsp Val Tyr Lys Pro Ile Ser Val 370 375 380 Leu Ser Gly Gly Glu Arg ValLys Val Ala Leu Thr Lys Val Phe Leu 385 390 395 400 Ser Glu Val Asn ThrLeu Val Leu Asp Glu Pro Thr Asn Phe Leu Asp 405 410 415 Met Glu Ala IleGlu Ala Phe Glu Ser Leu Leu Lys Glu Tyr Asn Gly 420 425 430 Ser Ile IlePhe Val Ser His Asp Arg Lys Phe Ile Glu Lys Val Ala 435 440 445 Thr ArgIle Met Thr Ile Asp Asn Lys Glu Ile Lys Ile Phe Asp Gly 450 455 460 ThrTyr Glu Gln Phe Lys Gln Ala Glu Lys Pro Thr Arg Asn Ile Lys 465 470 475480 Glu Asp Lys Lys Leu Leu Leu Glu Thr Lys Ile Thr Glu Val Leu Ser 485490 495 Arg Leu Ser Ile Glu Pro Ser Glu Glu Leu Glu Gln Glu Phe Gln Asn500 505 510 Leu Ile Asn Glu Lys Arg Asn Leu Asp Lys 515 520 50 560 PRTStaphylococcus sp. 50 Met Leu Lys Ile Asp Met Lys Asn Val Lys Lys TyrTyr Ala Asp Lys 1 5 10 15 Leu Ile Leu Asn Ile Lys Glu Leu Lys Ile TyrSer Gly Asp Lys Ile 20 25 30 Gly Ile Val Gly Lys Asn Gly Val Gly Lys ThrThr Leu Leu Lys Ile 35 40 45 Ile Lys Gly Leu Ile Glu Ile Asp Glu Gly AsnIle Ile Ile Ser Glu 50 55 60 Lys Thr Thr Ile Lys Tyr Ile Ser Gln Leu GluGlu Pro His Ser Lys 65 70 75 80 Ile Ile Asp Gly Lys Tyr Ala Ser Ile PheGln Val Glu Asn Lys Trp 85 90 95 Asn Asp Asn Met Ser Gly Gly Glu Lys ThrArg Phe Lys Leu Ala Glu 100 105 110 Gly Phe Gln Asp Gln Cys Ser Leu MetLeu Val Asp Glu Pro Thr Ser 115 120 125 Asn Leu Asp Ile Glu Gly Ile GluLeu Ile Thr Asn Thr Phe Lys Glu 130 135 140 Tyr Arg Asp Thr Phe Leu ValVal Ser His Asp Arg Ile Phe Leu Asp 145 150 155 160 Gln Val Cys Thr LysIle Phe Glu Ile Glu Asn Gly Tyr Ile Arg Glu 165 170 175 Phe Ile Gly AsnTyr Thr Asn Tyr Ile Glu Gln Lys Glu Met Leu Leu 180 185 190 Arg Lys GlnGln Glu Glu Tyr Glu Lys Tyr Asn Ser Lys Arg Lys Gln 195 200 205 Leu GluGln Ala Ile Lys Leu Lys Glu Asn Lys Ala Gln Gly Met Ile 210 215 220 LysPro Pro Ser Lys Thr Met Gly Thr Ser Glu Ser Arg Ile Trp Lys 225 230 235240 Met Gln His Ala Thr Lys Gln Lys Lys Met His Arg Asn Thr Lys Ser 245250 255 Leu Glu Thr Arg Ile Asp Lys Leu Asn His Val Glu Lys Ile Lys Glu260 265 270 Leu Pro Ser Ile Lys Met Asp Leu Pro Asn Arg Glu Gln Phe HisGly 275 280 285 Arg Asn Val Ile Ser Leu Lys Asn Leu Ser Ile Lys Phe AsnAsn Gln 290 295 300 Phe Leu Trp Arg Asp Ala Ser Phe Val Ile Lys Gly GlyGlu Lys Val 305 310 315 320 Ala Ile Ile Gly Asn Asn Gly Val Gly Lys ThrThr Leu Leu Lys Leu 325 330 335 Ile Leu Glu Lys Val Glu Ser Val Ile IleSer Pro Ser Val Lys Ile 340 345 350 Gly Tyr Val Ser Gln Asn Leu Asp ValLeu Gln Ser His Lys Ser Ile 355 360 365 Leu Glu Asn Val Met Ser Thr SerIle Gln Asp Glu Thr Ile Ala Arg 370 375 380 Ile Val Leu Ala Arg Leu HisPhe Tyr Arg Asn Asp Val His Lys Glu 385 390 395 400 Ile Asn Val Leu SerGly Gly Glu Gln Ile Lys Val Ala Phe Ala Lys 405 410 415 Leu Phe Val SerAsp Cys Asn Thr Leu Ile Leu Asp Glu Pro Thr Asn 420 425 430 Tyr Leu AspIle Asp Ala Val Glu Ala Leu Glu Glu Leu Leu Ile Thr 435 440 445 Tyr GluGly Val Val Leu Phe Ala Ser His Asp Lys Lys Phe Ile Gln 450 455 460 AsnLeu Ala Glu Gln Leu Leu Ile Ile Glu Asn Asn Lys Val Lys Lys 465 470 475480 Phe Glu Gly Thr Tyr Ile Glu Tyr Leu Lys Ile Lys Asp Lys Pro Lys 485490 495 Leu Asn Thr Asn Glu Lys Glu Leu Lys Glu Lys Lys Met Ile Leu Glu500 505 510 Met Gln Ile Ser Ser Leu Leu Ser Lys Ile Ser Met Glu Glu AsnGlu 515 520 525 Glu Lys Asn Lys Glu Leu Asp Glu Lys Tyr Lys Leu Lys LeuLys Glu 530 535 540 Leu Lys Ser Leu Asn Lys Asn Ile Met Lys Tyr Gly ProAsp Pro Asn 545 550 555 560 51 507 PRT Staphylococcus sp. 51 Met Asn PheTyr Leu Glu Glu Phe Asn Leu Ser Ile Pro Asp Ser Gly 1 5 10 15 Pro TyrGly Ile Thr Ser Ser Glu Asp Gly Lys Val Trp Phe Thr Gln 20 25 30 His LysAla Asn Lys Ile Ser Ser Leu Asp Gln Ser Gly Arg Ile Lys 35 40 45 Glu PheGlu Val Pro Thr Pro Asp Ala Lys Val Met Cys Leu Ile Val 50 55 60 Ser SerLeu Gly Asp Ile Trp Phe Thr Glu Asn Gly Ala Asn Lys Ile 65 70 75 80 GlyLys Leu Ser Lys Lys Gly Gly Phe Thr Glu Tyr Pro Leu Pro Gln 85 90 95 ProAsp Ser Gly Pro Tyr Gly Ile Thr Glu Gly Leu Asn Gly Asp Ile 100 105 110Trp Phe Thr Gln Leu Asn Gly Asp Arg Ile Gly Lys Leu Thr Ala Asp 115 120125 Gly Thr Ile Tyr Glu Tyr Asp Leu Pro Asn Lys Gly Ser Tyr Pro Ala 130135 140 Phe Ile Thr Leu Gly Ser Asp Asn Ala Leu Trp Phe Thr Glu Asn Gln145 150 155 160 Asn Asn Ser Ile Gly Arg Ile Thr Asn Thr Gly Lys Leu GluGlu Tyr 165 170 175 Pro Leu Pro Thr Asn Ala Ala Ala Pro Val Gly Ile ThrSer Gly Asn 180 185 190 Asp Gly Ala Leu Trp Phe Val Glu Ile Met Gly AsnLys Ile Gly Arg 195 200 205 Ile Thr Thr Thr Gly Glu Ile Ser Glu Tyr AspIle Pro Thr Pro Asn 210 215 220 Ala Arg Pro His Ala Ile Thr Ala Gly LysAsn Ser Glu Ile Trp Phe 225 230 235 240 Thr Glu Trp Gly Ala Asn Gln IleGly Arg Ile Thr Asn Asp Lys Thr 245 250 255 Ile Gln Glu Tyr Gln Leu GlnThr Glu Asn Ala Glu Pro His Gly Ile 260 265 270 Thr Phe Gly Lys Asp GlySer Val Trp Phe Ala Leu Lys Cys Lys Ile 275 280 285 Gly Lys Leu Asn LeuAsn Glu Met Lys Trp Gln Asn Gln Gln Gly Pro 290 295 300 Asn Pro Glu GluIle Tyr Pro Ile Glu Gly Asn Lys His Val Gln Phe 305 310 315 320 Ile LysPro Ser Ile Thr Lys Pro Asn Ile Leu Val Gly Glu Tyr Ser 325 330 335 TyrTyr Asp Ser Lys Asp Gly Glu Ser Phe Glu Ser Gln Val Leu Tyr 340 345 350His Tyr Glu Leu Ile Gly Asp Lys Leu Ile Leu Gly Lys Phe Cys Ser 355 360365 Ile Gly Pro Gly Thr Thr Phe Ile Met Asn Gly Ala Asn His Arg Met 370375 380 Asp Gly Ser Thr Phe Pro Phe Asn Leu Phe Gly Asn Gly Trp Glu Lys385 390 395 400 His Thr Pro Thr Leu Glu Asp Leu Pro Tyr Lys Gly Asn ThrGlu Ile 405 410 415 Gly Asn Asp Val Trp Ile Gly Arg Asp Val Thr Ile MetPro Gly Val 420 425 430 Lys Ile Gly Asn Gly Ala Ile Ile Ala Ala Lys SerVal Val Thr Lys 435 440 445 Asn Val Asp Pro Tyr Ser Val Val Gly Gly AsnPro Ser Arg Leu Ile 450 455 460 Lys Ile Arg Phe Ser Lys Glu Lys Ile AlaAla Leu Leu Lys Val Arg 465 470 475 480 Trp Trp Asp Leu Glu Ile Glu ThrIle Asn Glu Asn Ile Asp Cys Ile 485 490 495 Leu Asn Gly Asp Ile Lys LysVal Lys Arg Ser 500 505

What is claimed is:
 1. A purified polynucleotide consisting of anucleotide sequence selected from the group consisting of SEQ ID NO:1 ora fragment derived from SEQ ID NO:1 containing 15 to 40 contiguousnucleotides, SEQ ID NO:11, and SEQ ID NO:12.
 2. A polynucleotidecomprising the full length coding sequence of a Staphylococcusstreptogramin A and/or B resistant gene containing a polynucleotidesequence according to claim
 1. 3. A recombinant cell host comprising apolynucleotide sequence selected from the group consisting of SEQ IDNO:1 and SEQ ID NO:12.